Fusion protein having luminescence activity

ABSTRACT

The fusion protein comprising (1) a first region comprising the amino acid sequence of SEQ ID NO: 18 and (2) a second region comprising an amino acid sequence for a polypeptide containing at least one cysteine residue for binding to other useful compound via the thiol group can be modified by chemical modification, and thus has a high catalytic ability for a luminescence activity and is highly available for general purposes.

TECHNICAL FIELD

The present invention relates to a fusion protein, a polynucleotide, arecombinant vector, a transformant, a method for producing the fusionprotein, a complex of the fusion protein to which other useful compoundis bound, etc., which are described below.

BACKGROUND ART

Gaussia luciferase is a secretory enzyme produced from the deep-seacopepoda, Gaussia princeps, and the luminescence system catalyzed byGaussia luciferase is simple and the luminescence reaction is performedonly under the presence of oxygen and a substrate (a luciferin such as acoelenterazine, etc.).

By comparison with the luminescence reaction catalyzed by fireflyluciferase, the luminescence reaction catalyzed by Gaussia luciferase issimple and emits strong blue light. On the other hand, fireflyluciferase requires ATP and magnesium ions for the luminescencereaction. From this reason, Gaussia luciferase expects to be used invarious applications in the future.

Gaussia luciferase is a simple protein having the signal peptidesequence of 17 amino acid residues at the amino terminus for secretionand the catalytic domain consisting of 168 amino acid residues. Gaussialuciferase is a unique luciferase having 10 cysteine residues in amolecule, in which the content of cysteines is approximately 6% in thetotal amino acid. It has been reported that an intramolecular —S—S— bondis critical to retain the catalytic ability for the luminescenceactivity of Gaussia luciferase. By treatment of Gaussia luciferase withreducing agents such as mercaptoethanol, dithiothreitol, etc., theluminescence activity of Gaussia luciferase was lost completely (Inouye,S. & Sahara, Y. (2008) Biochem. Biophys. Res. Commun. 365, 96-101.).However, there is little information on the positions of theintramolecular —S—S— bond or free thiol groups in Gaussia luciferase.

By the chemical modification of Gaussia luciferase with a ligand usingthe intramolecular thiol groups of Gaussia luciferase, namely, byintroducing a ligand into Gaussia luciferase via thiol groups of aminoacid residue in Gaussia luciferase, the ligand-conjugated Gaussialuciferase can be obtained. Using the ligand-conjugated Gaussialuciferase and relying on the luminescence reaction of Gaussialuciferase, it is considered to detect a substance capable ofspecifically binding to the ligand. However, there is no report so faron such a case that Gaussia luciferase was directly conjugated with theligand by chemical modification.

The present inventors found that when a ligand is introduced intoGaussia luciferase via the thiol groups from the amino acids whichconstitute Gaussia luciferase, the catalytic ability of theligand-conjugated Gaussia luciferase for the luminescence activity ismarkedly reduced. The inventors also attempted to introduce a ligandinto Gaussia luciferase via amino groups or carboxyl groups, other thanthe thiol groups, derived from the amino acids which constitute Gaussialuciferase. However, the catalytic ability of Gaussia luciferase for theluminescence activity was likewise markedly reduced. As such, it wasimpossible to obtain the ligand-conjugated Gaussia luciferase bychemical modification while maintaining the original luminescenceintensity of Gaussia luciferase. This is considered to be because theprotein catalytic domains associated with the luminescence reaction ofGaussia luciferase would be affected by introducing the ligand via thethiol, amino or carboxyl groups derived from the amino acids in themolecule of Gaussia luciferase, and the luminescence reaction would beinhibited.

On the other hand, it is considered that when cysteine residues areintroduced into the molecule or at the amino or carboxyl terminus ofGaussia luciferase, the formation of correct intramolecular —S—S— bondsby refolding of a protein expressed in cells would be hindered.

The only ligand-conjugated Gaussia luciferase reported is biotinylatedGaussia luciferase modified with a biotinylation enzyme. This luciferasewas prepared by expressing the fused Gaussia luciferase gene having amodifiable biotin recognition sequence in Escherichia coli followed bybiotinylation with an enzyme present in E. coli (Verhaegen, M. &Christopoulos, T. K. (2002) Anal. Chem. 74, 4378-4385.). According to anenzymatic modification in living cells as in this method, however, it isdifficult to supply biotinylated Gaussia luciferase uniformly in largeamounts. Furthermore, according to this method the ligand which can beintroduced into Gaussia luciferase is biotin alone, but other ligandssuch as avidin, streptavidin, enzymes, antibodies, antigens, nucleicacids, polysaccharides, receptors or the like or fluorescent substances,etc. cannot be introduced into Gaussia luciferase. The ligand which canbe introduced is biotin alone and its application is limited.

DISCLOSURE OF THE INVENTION

Under the circumstances described above, a luciferase which can bemodified through chemical modification, has a high catalytic ability forthe luminescence activity and is highly available for general purposeshas been desired.

As a result of extensive studies to solve the foregoing problems, thepresent inventors found that a fusion protein comprising Gaussialuciferase and a polypeptide having at least one cysteine residue forbinding to other useful compound via the thiol group is a luciferasewhich can be modified by chemical modification and has high availabilityfor general purposes, while retaining the catalytic ability of Gaussialuciferase for the luminescence activity. Based on these findings, theinventors have continued further investigations and come to accomplishthe present invention.

More specifically, the present invention provides a fusion protein, apolynucleotide, a recombinant vector, a transformant, a method forproducing the fusion protein, a complex of the fusion protein and otheruseful compound, etc., which are described below.

[1] A fusion protein comprising:

(1) a first region selected from the group consisting of (a) to (d)below:

(a) a region consisting of the amino acid sequence of SEQ ID NO: 18;

(b) a region consisting of the amino acid sequence of SEQ ID NO: 18wherein 1 or more amino acids are deleted, substituted, inserted and/oradded and having a catalytic ability for a luminescence activity with aluciferin which is a substrate;

(c) a region consisting of an amino acid sequence having at least 70%homology to the amino acid sequence of SEQ ID NO: 18 and having acatalytic ability for a luminescence activity with a luciferin which isa substrate; and,

(d) a region consisting of an amino acid sequence encoded by apolynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 17 and having a catalytic ability fora luminescence activity with a luciferin which is a substrate; and,

(2) a second region consisting of an amino acid sequence for apolypeptide having at least one cysteine residue for binding to otheruseful compound via its thiol group.

[2] The fusion protein according to [1] above, wherein the second regionis selected from the group consisting of (e) to (h) below:

(e) a region consisting of the amino acid sequence of SEQ ID NO: 20;

(f) a region comprising the amino acid sequence of SEQ ID NO: 20 wherein1 or more amino acids are deleted, substituted, inserted and/or addedand having at least one cysteine residue for binding to other usefulcompound via the thiol group;

(g) a region comprising an amino acid sequence having at least 70%homology to the amino acid sequence of SEQ ID NO: 20 and having at leastone cysteine residue for binding to other useful compound via the thiolgroup; and,

(h) a region comprising an amino acid sequence encoded by apolynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 19 and having at least one cysteineresidue for binding to other useful compound via the thiol group.

[3] The fusion protein according to [2] above, wherein the second regionis selected from the group consisting of (e) to (h) below:

(e) a region consisting of the amino acid sequence of SEQ ID NO: 20;

(f) a region comprising the amino acid sequence of SEQ ID NO: 20 wherein1 to 3 amino acids are deleted, substituted, inserted and/or added andhaving at least one cysteine residue for binding to other usefulcompound via the thiol group;

(g) a region comprising an amino acid sequence having at least 90%homology to the amino acid sequence of SEQ ID NO: 20 and having at leastone cysteine residue for binding to other useful compound via the thiolgroup; and,

(h) a region comprising an amino acid sequence encoded by apolynucleotide which hybridizes under high stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 19 and having at least one cysteineresidue for binding to other useful compound via the thiol group.

[4] The fusion protein according to any one of [1] to [3] above, whereinthe first region is selected from the group consisting of (a) to (d)below:

(a) a region consisting of the amino acid sequence of SEQ ID NO: 18;

(b) a region consisting of the amino acid sequence of SEQ ID NO: 18wherein 1 to 10 amino acids are deleted, substituted, inserted and/oradded and having a catalytic ability for a luminescence activity with aluciferin which is a substrate;

(c) a region consisting of an amino acid sequence having at least 90%homology to the amino acid sequence of SEQ ID NO: 18 and having acatalytic ability for a luminescence activity with a luciferin which isa substrate; and,

(d) a region consisting of an amino acid sequence encoded by apolynucleotide which hybridizes under high stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 17 and having a catalytic ability fora luminescence activity with a luciferin which is a substrate.

[5] The fusion protein according to [4] above, wherein:

(1) the first region is a region consisting of the amino acid sequenceof SEQ ID NO: 18, and,

(2) the second region is a region consisting of the amino acid sequenceof SEQ ID NO: 20.

[6] The fusion protein according to any one of [1] to [5] above, furthercomprising an amino acid sequence for promoting translation and/or anamino acid sequence for purification.

[7] A fusion protein consisting of an amino acid sequence of SEQ ID NO:4, 6 or 8.

[8] A polynucleotide comprising a polynucleotide encoding the fusionprotein according to any one of [1] to [7] above.

[9] A polynucleotide comprising:

(1) a first coding sequence selected from the group consisting of (a) to(d) below:

(a) a coding sequence consisting of a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 17;

(b) a coding sequence consisting of a polynucleotide which hybridizesunder stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 17 and encodes a region having a catalytic ability for aluminescence activity with a luciferin which is a substrate;

(c) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18; and,

(d) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18 wherein 1 or moreamino acids are deleted, substituted, inserted and/or added and having acatalytic ability for a luminescence activity with a luciferin which isa substrate; and,

(2) a second coding sequence consisting of a polynucleotide encoding apolypeptide having at least one cysteine residue for binding to otheruseful compound via the thiol group.

[10] The polynucleotide according to [9] above, wherein the secondcoding sequence is selected from the group consisting of (e) to (h)below:

(e) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the nucleotide sequence of SEQ ID NO: 19;

(f) a coding sequence consisting of a polynucleotide which hybridizesunder stringent conditions to a polynucleotide complementary to anucleotide sequence consisting of the nucleotide sequence of SEQ ID NO:19 and encodes a region having at least one cysteine residue for bindingto other useful compound via the thiol group;

(g) a coding region consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20; and,

(h) a coding region consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20 wherein 1 or moreamino acids are deleted, substituted, inserted and/or added and havingat least one cysteine residue for binding to other useful compound viathe thiol group.

[11] The polynucleotide according to [10] above, wherein the secondcoding sequence is selected from the group consisting of (e) to (h)below:

(e) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the nucleotide sequence of SEQ ID NO: 19;

(f) a coding sequence consisting of a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 19 and encodes a region having at least one cysteine residue forbinding to other useful compound via the thiol group;

(g) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20; and,

(h) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20 wherein 1 to 3amino acids are deleted, substituted, inserted and/or added and havingat least one cysteine residue for binding to other useful compound viathe thiol group.

[12] The polynucleotide according to any one of [9] to [11] above,wherein the first coding sequence is selected from the group consistingof (a) to (d) below:

(a) a coding sequence consisting of a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 17;

(b) a coding sequence consisting of a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 17 and encodes a region having a catalytic ability for aluminescence activity with a luciferin which is a substrate;

(c) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18; and,

(d) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18 wherein 1 to 10amino acids are deleted, substituted, inserted and/or added and having acatalytic ability for a luminescence activity with a luciferin which isa substrate.

[13] The polynucleotide according to [12] above, wherein:

(1) the first coding sequence is a coding sequence consisting of apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 17;and,

(2) the second coding sequence is a coding sequence consisting of apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19.

[14] A polynucleotide comprising a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 3, 5 or 7.

[15] A recombinant vector comprising the polynucleotide according to anyone of [8] to [14] above.

[16] A transformant transformed with the recombinant vector according to[15] above.

[17] A method for producing the fusion protein according to any one of[1] to [7] above, which comprises culturing the transformant of [16]above and producing the fusion protein according to any one of [1] to[7] above.

[18] A complex comprising the fusion protein according to any one of [1]to [7] above and other useful compound bound to the fusion protein viathe thiol group of the cysteine residue in the second region.

[19] The complex according to [18] above, wherein other useful compoundis a fluorescent substance and/or a ligand specific to an analyte.

[20] A kit comprising the fusion protein according to any one of [1] to[7] above.

[21] A kit comprising the polynucleotide according to any one of [8] to[14] above, the recombinant vector according to [15] above or thetransformant according to [16] above.

[22] A kit comprising the complex of [18] or [19] above.

[23] The kit according to any one of [20] to [22] above, furthercomprising a luciferin.

[24] The kit according to [23] above, wherein the luciferin is acoelenterazine analogue.

[25] The kit according to [24] above, wherein the coelenterazineanalogue is coelenterazine.

[26] A method for performing a luminescence reaction, which comprisescontacting the fusion protein according to any one of [1] to [7] aboveor the complex according to [18] or [19] above with a luciferin.

[27] A method for analyzing a physiological function or determining anenzyme activity, which comprises performing bioluminescence resonanceenergy transfer (BRET) using the fusion protein according to any one of[1] to [7] above or the complex according to [18] or [19] above as adonor protein.

[28] A method for determining a substance specific to the ligand, whichcomprises using the complex according to [19] above.

The fusion protein of the present invention is a luciferase which can bemodified by chemical modification and is highly available for generalpurposes. In a preferred embodiment of the present invention, the fusionprotein retains the catalytic ability of Gaussia luciferase for theluminescence activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing the expression vector pPICZα-hgLinkerhaving a hinge sequence and a multicloning site used in the presentinvention.

FIG. 2 is a schematic view showing the expression vector pPICZα-hgGL-Hfor hg-Gaussia luciferase to express in yeast, which is used in thepresent invention.

FIG. 3 is a schematic view showing the expression vector pCold-hgGL forhg-Gaussia luciferase to express in E. coli, which is used in thepresent invention.

FIG. 4 is a schematic view showing the expression vector pCold-hgA-GLfor hgA-Gaussia luciferase to express in E. coli, which is used in thepresent invention.

FIG. 5 shows the results of SDS-PAGE analysis in the process ofpurification from the soluble fractions of hg-Gaussia luciferase. Lane1: protein molecular weight marker (Tefco), Lane 2: supernatant obtainedby centrifuging the ultrasonicated lysate of the transformant frompCold-hg-Gaussia luciferase-expressed E. coli at 12,000 g for 20 minutes(protein concentration, 18.5 μg), Lane 3: fraction eluted from anickel-chelate column (protein concentration, 7.4 μg), Lane 4: fractioneluted from a butyl column (protein concentration, 1.1 μg).

FIG. 6 shows the results of SDS-PAGE analysis in the process ofpurification from the soluble fractions of hgA-Gaussia luciferase. Lane1: protein molecular weight marker (Tefco), Lane 2: supernatant obtainedby centrifuging the ultrasonicated lysate of the transformant frompCold-hgA-Gaussia luciferase-expressed E. coli at 12,000 g for 20minutes (protein concentration, 17.7 μg), Lane 3: fraction eluted from anickel-chelate column (protein concentration, 14.5 μg), Lane 4: fractioneluted from a butyl column (protein concentration, 0.72 μg).

FIG. 7 shows the relationship between the concentration and luminescenceintensity of biotinylated hg-Gaussia luciferase.

FIG. 8 shows the standard curve of AFP obtained using biotinylatedhg-Gaussia luciferase, wherein the solid line represents the backgroundconcentration, indicating a mean value+3SD when the concentration of AFPis 0 ng/ml.

FIG. 9 shows the relationship between the concentration and luminescenceintensity of biotinylated hgA-Gaussia luciferase.

FIG. 10 shows the standard curve of AFP obtained using biotinylatedhgA-Gaussia luciferase, wherein the solid line represents the backgroundconcentration, indicating a mean value+3SD when the concentration of AFPis 0 ng/ml.

FIG. 11 shows the relationship between the concentration of hgA-GL-Ab1D5and the luminescence intensity.

FIG. 12 shows the standard curve of AFP obtained using hgA-GL-Ab1D5,wherein the solid line represents the background concentration,indicating a mean value+3SD when the concentration of AFP is 0 ng/ml.

FIG. 13 shows the bioluminescence energy transfer byfluorescence-labeled Gaussia luciferase, wherein the dotted linerepresents the luminescence spectra of Gaussia luciferase and the solidline represents the luminescence spectra of fluorescence-labeled Gaussialuciferase.

FIG. 14 shows the expression vector (pCold-GL-AQ-S142C) for Gaussialuciferase-apoaequorin-S142C and the fusion protein expressed; (a) is aschematic view of the expression vector and (b) is a schematic view ofthe amino acid sequence for the fusion protein expressed.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

1. Fusion Protein of the Invention

The fusion protein of the present invention is a fusion proteincomprising (1) a first region selected from the group consisting of aregion consisting of the amino acid sequence of SEQ ID NO: 18 and aregion having substantially the same activity or function as the regionconsisting of the amino acid sequence of SEQ ID NO: 18, and (2) a secondregion consisting of an amino acid sequence for a polypeptide having atleast one cysteine residue for binding to other useful compound via thethiol group.

In the fusion protein of the present invention, the first region and thesecond region may be arranged either in the order of “the N terminus-thefirst region-the second region-the C terminus” or in the order of “the Nterminus-the second region-the first region-the C terminus.” In thefusion protein in a preferred embodiment of the present invention, thefirst region and the second region are arranged in the order of “the Nterminus-the first region-the second region-the C terminus.”

(1) First Region

The first region is intended to mean a region consisting of the aminoacid sequence of SEQ ID NO: 18 or a region having substantially the sameactivity or function as the region consisting of the amino acid sequenceof SEQ ID NO: 18.

The term substantially the same activity or function is intended to meana catalytic ability for a luminescence activity with a luciferin (e.g.,a coelenterazine analogue) which is a substrate (hereinafter sometimesreferred to as “luminescence activity”), namely, an activity ofcatalyzing the reaction where luciferin (e.g., coelenterazine analogue)is oxidized by oxygen molecules to form oxyluciferin in its excitedstate. The oxyluciferin formed in the excited state emits visible lightand turns to the ground state.

The catalytic ability for the luminescence activity described above canbe determined by the method described in, e.g., Inouye, S. & Sahara, Y.(2008) Biochem. Biophys. Res. Commun. 365, 96-101, etc. Specifically,the fusion protein of the present invention is mixed with a luciferin toinitiate a luminescence reaction and the catalytic ability for theluminescence activity can be determined using an apparatus for measuringluminescence, e.g., Luminescencer-PSN AB2200 (manufactured by Atto),Centro 960 luminometer (manufactured by Berthold), etc.

The luciferin used in the present invention may be any luciferin as longas it serves as a substrate for the fusion protein of the presentinvention. Specifically, the luciferin used in the present inventionincludes a coelenterazine analogue.

As used herein, the coelenterazine analogue is intended to meancoelenterazine and a coelenterazine derivative. Examples of thecoelenterazine derivative include h-coelenterazine, hcp-coelenterazine,cp-coelenterazine, f-coelenterazine, fcp-coelenterazine,n-coelenterazine, Bis-coelenterazine, MeO-coelenterazine,e-coelenterazine, cl-coelenterazine, ch-coelenterazine, and the like. Ofthese coelenterazine analogues, coelenterazine is particularly preferredin the present invention. These coelenterazine analogues may besynthesized by publicly known methods or may also be commerciallyavailable.

The coelenterazine analogues can be synthesized by the methods describedin, e.g., Shimomura et al. (1988) Biochem. J. 251, 405-410, Shimomura etal. (1989) Biochem. J. 261, 913-920, Shimomura et al. (1990) Biochem. J.270, 309-312, etc. or modifications thereof.

The coelenterazine analogues which are commercially available include,for example, coelenterazine and h-coelenterazine manufactured by ChissoCorporation; hcp-coelenterazine, cp-coelenterazine, f-coelenterazine,fcp-coelenterazine and n-coelenterazine manufactured by Sigma Inc., andthe like.

Specifically, the first region is selected from the group consisting of(a) to (d) below:

(a) a region consisting of the amino acid sequence of SEQ ID NO: 18;

(b) a region consisting of the amino acid sequence of SEQ ID NO: 18wherein 1 or more amino acids are deleted, substituted, inserted and/oradded and having a catalytic ability for a luminescence activity with aluciferin which is a substrate;

(c) a region consisting of an amino acid sequence having at least 70%homology to the amino acid sequence of SEQ ID NO: 18 and having acatalytic ability for a luminescence activity with a luciferin which isa substrate; and,

(d) a region consisting of an amino acid sequence encoded by apolynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 17 and having a catalytic ability fora luminescence activity with a luciferin which is a substrate.

In the first region, the “deletion, substitution, insertion and/oraddition of one or more amino acid residues” means that one or aplurality of amino acid residues are deleted, substituted, insertedand/or added at an optional position(s) in the same sequence and at aposition(s) in one or a plurality of amino acid sequences.

Examples of amino acid residues which are mutually substitutable aregiven below. Amino acid residues in the same group are mutuallysubstitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine,t-butylalanine and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid and 2-aminosuberic acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline and 4-hydroxyproline;

Group F: serine, threonine and homoserine; and,

Group G: phenylalanine and tyrosine.

In the first region, the range of “1 or more” in “the amino acidsequence in which 1 or more amino acids are deleted, substituted,inserted and/or added” is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to2, and 1. In general, the less the number of amino acids deleted,substituted, inserted or added, the more preferable. In the deletion,substitution, insertion and addition of the amino acid residuesdescribed above, two or more may occur concurrently. Such domains can beacquired site-directed mutagenesis described in Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press (2001); Ausbel F. M. et al., Current Protocolsin Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997);Nuc. Acids. Res., 10, 6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409(1982); Gene, 34, 315 (1985); Nuc. Acids. Res., 13, 4431 (1985); Proc.Natl. Acad. Sci. USA, 82, 488 (1985); etc.

In the first region, the range of “at least 70%” in the “amino acidsequence having at least 70% homology” is, for example, 70% or more, 75%or more, 80% or more, 85% or more, 88% or more, 90% or more, 91% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more,99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% ormore, 99.8% or more, or 99.9% or more. In general, the numerical valueof the homology described above is more preferable as the number becomeslarger. The homology of nucleotide sequences or amino acid sequences canbe determined using a sequencing program such as BLAST (see, e.g.,Altzchul, S. F. et al., J. Mol. Biol., 215, 403 (1990), etc.) or thelike. When BLAST is used, the default parameters for the respectiveprograms are employed.

In the first region, the “polynucleotide which hybridizes understringent conditions” is intended to mean a polynucleotide (e.g., DNA)which is obtained by, for example, colony hybridization, plaquehybridization or Southern hybridization using as a probe all or part ofthe polynucleotide consisting of a nucleotide sequence complementary tothe nucleotide sequence of SEQ ID NO: 17 or the polynucleotide encodingthe amino acid sequence of SEQ ID NO: 18. Specific examples include apolynucleotide which can be identified by performing hybridization at65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on whichthe polynucleotide from a colony or plaque is immobilized, then washingthe filter at 65° C. with an SSC (saline-sodium citrate) solution havinga concentration of 0.1 to 2 times (1×SSC solution is composed of 150mmol/L sodium chloride and 15 mmol/L sodium citrate).

Hybridization may be performed in accordance with modifications of themethods described in laboratory manuals, e.g., Sambrook, J. et al.:Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press (2001); Ausbel F. M. et al., Current Protocolsin Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997);Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, Apractical Approach, Second Edition, Oxford University Press (1995); etc.

As used herein, the “stringent conditions” may be any of low stringentconditions, moderate stringent conditions or high stringent conditions.The “low-stringent conditions” are, for example, conditions of 5×SSC,5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 32° C.The “moderate stringent conditions” are, for example, conditions of5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and42° C. The “high-stringent conditions” are, for example, 5×SSC,5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50° C.The more stringent the conditions are, the higher the complementarityrequired for double-strand formation. Specifically, for example, underthese conditions, a polynucleotide (e.g., DNA) of higher homology isexpected to be obtained efficiently as the temperature becomes higher,although multiple factors are involved in hybridization stringency,including temperature, probe concentration, probe length, ionicstrength, time, salt concentration, etc. One skilled in the art mayachieve a similar stringency by appropriately choosing these factors.

When a commercially available kit is used for hybridization, forexample, Alkphos Direct Labeling Reagents (manufactured by AmershamPharmacia) can be used. In this case, according to the attachedprotocol, a membrane is incubated with a labeled probe overnight, themembrane is washed with a primary wash buffer containing 0.1% (w/v) SDSunder conditions at 55° C. and then the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having the homology of approximately 60% or more, 65%or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% ormore, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more,99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8%, or99.9% or more, to the polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 17 or the polynucleotide encoding the amino acidsequence of SEQ ID NO: 18. The homology of nucleotide sequences or aminoacid sequences can be determined using the method described above.

In a preferred embodiment of the present invention, the first region isselected from the group consisting of (a) to (d) below:

(a) a region consisting of the amino acid sequence of SEQ ID NO: 18;

(b) a region consisting of the amino acid sequence of SEQ ID NO: 18wherein 1 to 10 amino acids are deleted, substituted, inserted and/oradded and having a catalytic ability for a luminescence activity with aluciferin which is a substrate;

(c) a region consisting of an amino acid sequence having at least 90%homology to the amino acid sequence of SEQ ID NO: 18 and having acatalytic ability for a luminescence activity with a luciferin which isa substrate; and,

(d) a region consisting of an amino acid sequence encoded by apolynucleotide which hybridizes under high stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 17 and having a catalytic ability fora luminescence activity with a luciferin which is a substrate.

More preferably, the first region is a region consisting of the aminoacid sequence of SEQ ID NO: 18.

(2) Second Region

The second region is a region consisting of an amino acid sequence for apolypeptide having at least one cysteine residue for binding to otheruseful compound via the thiol group. The term “1 or more” in the term“having at least one cysteine residue for binding to other usefulcompound via the thiol group” is used to mean, for example, 1, 2 or 3,preferably 1 or 2 and more preferably 1. Other useful compound can beintroduced into the fusion protein of the present invention by chemicalmodification via the thiol group derived from cysteine residuescontained in the second region. In a preferred embodiment of the presentinvention, other useful compound can be introduced into the fusionprotein of the present invention by chemical modification via the thiolgroup derived from cysteine residues contained in the second region,without any significant loss of the catalytic ability for theluminescence activity in the first region.

In the second region, the polypeptide has a length of, e.g., 2 to 40amino acids, preferably 5 to 35 amino acids, more preferably 7 to 30amino acids, much more preferably 10 to 20 amino acids and mostpreferably 15 amino acids.

Other useful compounds for binding to the second region includefluorescent substances, ligands specific to analytes (e.g., biotin,biotin-conjugated proteins, enzymes, substrates, antibodies, antigens,nucleic acids, polysaccharides, receptors or compounds capable ofbinding thereto, etc.), and the like.

The “fluorescent substances” and “ligands” are those described below.

In some embodiments of the present invention, the second region isselected from the group consisting of (e) to (h) below:

(e) a region consisting of the amino acid sequence of SEQ ID NO: 20;

(f) a region comprising the amino acid sequence of SEQ ID NO: 20 wherein1 or more amino acids are deleted, substituted, inserted and/or addedand having at least one cysteine residue for binding to other usefulcompound via the thiol group;

(g) a region comprising an amino acid sequence having at least 70%homology to the amino acid sequence of SEQ ID NO: 20 and having at leastone cysteine residue for binding to other useful compound via the thiolgroup; and,

(h) a region comprising an amino acid sequence encoded by apolynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 19 and having at least one cysteineresidue for binding to other useful compound via the thiol group.

In the second region, “the deletion, substitution, insertion and/oraddition of one or more amino acid residues” means that one or aplurality of amino acid residues are deleted, substituted, insertedand/or added at an optional position(s) in the same sequence and at aposition(s) in one or a plurality of amino acid sequences.

Examples of amino acid residues which are mutually substitutable aregiven below. Amino acid residues in the same group are mutuallysubstitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine,t-butylalanine and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid and 2-aminosuberic acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline and 4-hydroxyproline;

Group F: serine, threonine and homoserine; and,

Group G: phenylalanine and tyrosine.

In the second region, the range of “1 or more” in “the amino acidsequence in which 1 or more amino acids are deleted, substituted,inserted and/or added” is, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2,and 1. In general, the less the number of amino acids deleted,substituted, inserted or added, the more preferable. In the deletion,substitution, insertion and addition of the amino acid residuesdescribed above, two or more may occur concurrently. Such domains can beacquired site-directed mutagenesis described in Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press (2001); Ausbel F. M. et al., Current Protocolsin Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997);Nuc. Acids. Res., 10, 6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409(1982); Gene, 34, 315 (1985); Nuc. Acids. Res., 13, 4431 (1985); Proc.Natl. Acad. Sci. USA, 82, 488 (1985); etc.

In the second region, the range of “at least 70%” in the “amino acidsequence having at least 70% homology” is, for example, 70% or more, 75%or more, 80% or more, 85% or more, 88% or more, 90% or more, 91% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more,99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% ormore, 99.8% or more, or 99.9% or more. In general, the numerical valueof the homology described above is more preferable as the number becomeslarger. The homology of nucleotide sequences or amino acid sequences canbe determined using a sequencing program such as BLAST (see, e.g.,Altzchul, S. F. et al., J. Mol. Biol., 215, 403 (1990), etc.) or thelike. When BLAST is used, the default parameters for the respectiveprograms are employed.

In the second region, the “polynucleotide which hybridizes understringent conditions” is intended to mean a polynucleotide (e.g., DNA)which is obtained by, for example, colony hybridization, plaquehybridization or Southern hybridization using as a probe all or part ofthe polynucleotide consisting of a nucleotide sequence complementary tothe nucleotide sequence of SEQ ID NO: 19 or the polynucleotide encodingthe amino acid sequence of SEQ ID NO: 20. Specific examples include apolynucleotide which can be identified by performing hybridization at65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on whichthe polynucleotide from a colony or plaque is immobilized, then washingthe filter at 65° C. with an SSC (saline-sodium citrate) solution havinga concentration of 0.1 to 2 times (1×SSC solution is composed of 150mmol/L sodium chloride and 15 mmol/L sodium citrate).

Hybridization may be performed in accordance with modifications of themethods described in laboratory manuals, e.g., Sambrook, J. et al.:Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press (2001); Ausbel F. M. et al., Current Protocolsin Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997);Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, Apractical Approach, Second Edition, Oxford University Press (1995); etc.

As used herein, the “stringent conditions” may be any of low stringentconditions, moderate stringent conditions or high stringent conditions.The “low-stringent conditions” are, for example, conditions of 5×SSC,5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 32° C.The “moderate stringent conditions” are, for example, conditions of5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and42° C. The “high-stringent conditions” are, for example, 5×SSC,5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50° C.The more stringent the conditions are, the higher the complementarityrequired for double-strand formation. Specifically, for example, underthese conditions, a polynucleotide (e.g., DNA) of higher homology isexpected to be obtained efficiently as the temperature becomes higher,although multiple factors are involved in hybridization stringency,including temperature, probe concentration, probe length, ionicstrength, time, salt concentration, etc. One skilled in the art mayachieve a similar stringency by appropriately choosing these factors.

When a commercially available kit is used for hybridization, forexample, Alkphos Direct Labeling Reagents (manufactured by AmershamPharmacia) can be used. In this case, according to the attachedprotocol, a membrane is incubated with a labeled probe overnight, themembrane is washed with a primary wash buffer containing 0.1% (w/v) SDSunder conditions at 55° C. and then the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having the homology of approximately 60% or more, 65%or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% ormore, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more,99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8%, or99.9% or more, to the polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 19 or the polynucleotide encoding the amino acidsequence of SEQ ID NO: 20. The homology of nucleotide sequences or aminoacid sequences can be determined using the method described above.

In a preferred embodiment of the present invention, the second region isselected from the group consisting of (e) to (h) below:

(e) a region consisting of the amino acid sequence of SEQ ID NO: 20;

(f) a region comprising the amino acid sequence of SEQ ID NO: 20 wherein1 to 3 amino acids are deleted, substituted, inserted and/or added andhaving at least one cysteine residue for binding to other usefulcompound via the thiol group;

(g) a region comprising an amino acid sequence having at least 90%homology to the amino acid sequence of SEQ ID NO: 20 and having at leastone cysteine residue for binding to other useful compound via the thiolgroup; and,

(h) a region comprising an amino acid sequence encoded by apolynucleotide which hybridizes under high stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 19 and having at least one cysteineresidue for binding to other useful compound via the thiol group.

In a more preferred embodiment of the present invention, the secondregion is a region consisting of the amino acid sequence of SEQ ID NO:20.

In a preferred embodiment of the present invention, the fusion proteinis a fusion protein comprising:

(1) a first region consisting of the amino acid sequence of SEQ ID NO:18 and (2) a second region consisting of the amino acid sequence of SEQID NO: 20.

In a more preferred embodiment of the present invention, the fusionprotein includes, for example, a fusion protein consisting of the aminoacid sequence of SEQ ID NO: 4, 6 or 8.

The fusion protein of the present invention may further contain anadditional peptide sequence at the N terminus and/or C terminus,preferably at the N terminus, as in the amino acid sequences of SEQ IDNOS: 4, 6 and 8. The additional peptide sequence includes, for example,at least one peptide sequence selected from the group consisting of apeptide sequence for promoting translation, a peptide sequence forpurification, a secretory signal peptide sequence, a peptide sequencefor expressing the fusion protein of the present invention as a solubleprotein, an epitope sequence capable of recognizing an antibody, etc.The additional peptide sequence is preferably a peptide sequence forpurification and/or a secretory signal peptide sequence. In a still morepreferred embodiment of the present invention, the additional peptidesequence is at least one sequence selected from the group consisting ofa peptide sequence for purification, a secretory signal peptide sequenceand a peptide sequence for expressing the fusion protein of the presentinvention as a soluble protein.

The fusion protein of the present invention may further contain a linkersequence for restriction enzyme sites, as in the amino acid sequences ofSEQ ID NOS: 4, 6 and 8.

Peptide sequences used in the art can be employed as the peptidesequence for promoting translation. Examples of the peptide sequence forpromoting translation are a TEE sequence, and the like.

Peptide sequences employed in the art can be used as the peptidesequence for purification. The peptide sequence for purificationincludes, for example, a histidine tag sequence having a consecutiveamino acid sequence of at least 4 and preferably at least 6 histidineresidues, an amino acid sequence with a binding domain of glutathioneS-transferase into glutathione, the amino acid sequence of Protein A, anavidin tag sequence, etc.

The secretory signal peptide is intended to mean a peptide region whichhas the role of transporting a polypeptide bound to the secretory signalpeptide across a cell membrane. Amino acid sequences of such secretorysignal peptides and nucleotide sequences encoding the same are wellknown in the art and reported (see, e.g., von Heijine, G., Biochim.Biophys. Acta, 947: 307-333 (1988); von Heijine, G., J. Membr. Biol.,115, 195-201 (1990); etc.). Specific examples of secretory signalpeptides include the secretory signal peptide from the outer membraneprotein A of E. coli (OmpA) (Ghrayeb, J. et al., (1984) EMBO J.3:2437-2442), the secretory signal peptide from cholera toxin obtainedfrom Vibrio cholerae, etc.

The peptide used to express the fusion protein of the present inventionas a soluble protein includes, for example, polypeptides represented byformula (Z)_(n). The amino acid sequences for the polypeptidesrepresented by formula (Z)_(n) and the nucleic acid sequences encodingthe same are described in, e.g., JPA KOKAI No. 2008-99669.

As the linker sequences for restriction enzyme sites, peptide sequencesused in the art can be employed.

In the fusion protein of the present invention in which the first regionand the second region are arranged in the order of “the N terminus-thefirst region-the second region-the C terminus,” the length of theportion between the first region and the C terminus except for the firstregion is, for example, 4 to 50 amino acids, preferably 7 to 45 aminoacids, more preferably 14 to 43 amino acids, much more preferably 17 to40 amino acids and most preferably 21 to 36 amino acids.

In the fusion protein of the present invention in which the first regionand the second region are arranged in the order of “the N terminus-thesecond region-the first region-the C terminus,” the length of theportion between the first region and the N terminus except for the firstregion is, for example, 4 to 50 amino acids, preferably 7 to 45 aminoacids, more preferably 14 to 43 amino acids, much more preferably 17 to40 amino acids and most preferably 21 to 36 amino acids.

The method for acquiring the fusion protein of the invention is notparticularly limited. The fusion protein of the invention may be afusion protein synthesized by chemical synthesis, or a recombinantprotein produced by a genetic engineering technique. If the fusionprotein of the invention is to be chemically synthesized, synthesis maybe carried out by, for example, the Fmoc (fluorenylmethyloxycarbonyl)method or the tBoc (t-butyloxycarbonyl) method. In addition, peptidesynthesizers available from, for example, Advanced ChemTech,PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega,PerSeptive, Shimadzu Corporation, etc. may also be used for chemicalsynthesis. If the fusion protein of the invention is to be produced by agenetic engineering technique, the fusion protein may be produced by aconventional genetic recombination technique. More specifically, thefusion protein of the invention may be produced by inserting apolynucleotide (e.g., DNA) encoding the fusion protein of the inventioninto a suitable expression system. The polynucleotide encoding thefusion protein of the invention and expression of the fusion protein ofthe invention in an expression system will be later described.

2. Polynucleotide of the Invention

The present invention also provides a polynucleotide encoding the fusionprotein of the invention described above. The polynucleotide of theinvention may be any polynucleotide as long as it has a nucleotidesequence encoding the fusion protein of the invention, although a DNA ispreferred. Examples of the DNA include genomic DNA, genomic DNA library,cellular or tissue cDNA, cellular or tissue cDNA library, synthetic DNA,etc. Vectors used in the libraries are not particularly limited and maybe any of bacteriophages, plasmids, cosmids, phagemids, etc. Also, thesevectors may be amplified directly by a reverse transcription polymerasechain reaction (hereinafter abbreviated as RT-PCR) using the total RNAor mRNA fraction prepared from the cell or tissue described above.

Specifically, the polynucleotide of the present invention includes apolynucleotide comprising:

(1) a first coding sequence selected from the group consisting of (a) to(d) below:

(a) a coding sequence consisting of a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 17;

(b) a coding sequence consisting of a polynucleotide which hybridizesunder stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 17 and encodes a region having a catalytic ability for aluminescence activity with a luciferin which is a substrate;

(c) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18; and,

(d) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18 wherein 1 or moreamino acids are deleted, substituted, inserted and/or added and having acatalytic ability for a luminescence activity with a luciferin which isa substrate; and,

(2) a second coding sequence consisting of a polynucleotide encoding apolypeptide having at least one cysteine residue for binding to otheruseful compound via the thiol group.

Preferably, the second coding sequence described above is selected fromthe group consisting of (e) to (h) below:

(e) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the nucleotide sequence of SEQ ID NO: 19;

(f) a coding sequence consisting of a polynucleotide which hybridizesunder stringent conditions to a polynucleotide complementary to anucleotide sequence consisting of the nucleotide sequence of SEQ ID NO:19 and encodes a region having at least one cysteine residue for bindingto other useful compound via the thiol group;

(g) a coding region consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20; and,

(h) a coding region consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20 wherein 1 or moreamino acids are deleted, substituted, inserted and/or added and havingat least one cysteine residue for binding to other useful compound viathe thiol group.

As used herein, the “polynucleotide which hybridizes under stringentconditions” in the first and second coding sequences refers to apolynucleotide (e.g., DNA) which is obtained by, for example, colonyhybridization, plaque hybridization or Southern hybridization using as aprobe all or part of the polynucleotide consisting of a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 17 or 19or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 18or 20. Specific examples are polynucleotides which can be identified byperforming hybridization at 65° C. in the presence of 0.7 to 1.0 mol/LNaCl using a filter on which a polynucleotide derived from a colony orplaque is immobilized, then washing the filter at 65° C. with an SSC(saline-sodium citrate) solution having a concentration in a range of0.1 to 2 times (a 1-fold SSC solution is composed of 150 mmol/L ofsodium chloride and 15 mmol/L of sodium citrate).

Hybridization can be carried out based on the methods described inlaboratory manuals such as Sambrook J. et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press(2001); Ausbel F. M. et al., Current Protocols in Molecular Biology,Supplement 1-38, John Wiley and Sons (1987-1997); Glover D. M. and HamesB. D., DNA Cloning 1: Core Techniques, A Practical Approach, SecondEdition, Oxford University Press (1995); etc.

As used herein, the “stringent conditions” may be any of low stringentconditions, moderate stringent conditions or high stringent conditions.The “low-stringent conditions” are, for example, conditions of 5×SSC,5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 32° C.The “moderate stringent conditions” are, for example, conditions of5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and42° C. The “high-stringent conditions” are, for example, 5×SSC,5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50° C.The more stringent the conditions are, the higher the complementarityrequired for double-strand formation. Specifically, for example, underthese conditions, a polynucleotide (e.g., DNA) of higher homology isexpected to be obtained efficiently as the temperature becomes higher,although multiple factors are involved in hybridization stringency,including temperature, probe concentration, probe length, ionicstrength, time, salt concentration, etc. One skilled in the art mayachieve a similar stringency by appropriately choosing these factors.

When a commercially available kit is used for hybridization, forexample, Alkphos Direct Labeling Reagents (manufactured by AmershamPharmacia) can be used. In this case, according to the attachedprotocol, a membrane is incubated with a labeled probe overnight, themembrane is washed with a primary wash buffer containing 0.1% (w/v) SDSunder conditions at 55° C. and then the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having the homology of approximately 60% or more, 65%or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% ormore, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more,99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8%, or99.9% or more, to the polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 17 or 19, or the polynucleotide encoding theamino acid sequence of SEQ ID NO: 18 or 20. The homology of nucleotidesequences or amino acid sequences can be determined using the methoddescribed above.

In the first and second coding sequences, “the amino acid sequence inwhich 1 or more amino acids are deleted, substituted, inserted and/oradded” is the same as explained for the first and second regions,respectively.

A polynucleotide encoding a region having a given amino acid sequence,in which one or more amino acids are deleted, substituted, insertedand/or added, can be obtained by using a site-specific mutagenesistechnique (see, e.g., Gotoh, T. et al., Gene 152, 271-275 (1995);Zoller, M. J., and Smith, M., Methods Enzymol. 100, 468-500 (1983);Kramer, W. et al., Nucleic Acids Res. 12, 9441-9456 (1984); Kramer W,and Fritz H. J., Methods. Enzymol. 154, 350-367 (1987); Kunkel, T. A.,Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985); Kunkel, Methods Enzymol.85, 2763-2766 (1988); etc.), the methods utilizing amber mutation (see,e.g., the gapped duplex method, Nucleic Acids Res., 12, 9441-9456(1984), etc.), etc.

Alternatively, a mutation can be introduced into the polynucleotide bymeans of a polymerase chain reaction (PCR) using a set of primersbearing on the respective 5′ ends a sequence in which the targetmutation (deletion, addition, substitution and/or insertion) has beenintroduced (see, e.g., Ho, S. N. et al., Gene, 77, 51 (1989), etc.).

Also, a polynucleotide encoding a partial protein fragment, which is onetype of deletion mutant, can be obtained using as the primers anoligonucleotide having a sequence which matches the nucleotide sequenceat the 5′ end of the region encoding the partial fragment to be producedin the polynucleotide encoding the target protein and an oligonucleotidehaving a sequence complementary to the nucleotide sequence at the 3′ endthereof, and performing PCR in which the polynucleotide encoding thetarget protein serves as a template.

In the polynucleotide in a preferred embodiment of the presentinvention, the first coding sequence is selected from the groupconsisting of (a) to (d) below:

(a) a coding sequence consisting of a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 17;

(b) a coding sequence consisting of a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 17 and encodes a region having a catalytic ability for aluminescence activity with a luciferin which is a substrate;

(c) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18; and,

(d) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 18 wherein 1 to 10amino acids are deleted, substituted, inserted and/or added and having acatalytic ability for a luminescence activity with a luciferin which isa substrate.

In the polynucleotide in a preferred embodiment of the presentinvention, the second coding sequence is selected from the groupconsisting of (e) to (h) below:

(e) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the nucleotide sequence of SEQ ID NO: 19;

(f) a coding sequence consisting of a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 19 and encodes a region having at least one cysteine residue forbinding to other useful compound via the thiol group;

(g) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20; and,

(h) a coding sequence consisting of a polynucleotide encoding a regionconsisting of the amino acid sequence of SEQ ID NO: 20 wherein 1 to 3amino acids are deleted, substituted, inserted and/or added and havingat least one cysteine residue for binding to other useful compound viathe thiol group.

In a more preferred embodiment of the present invention, thepolynucleotide is a polynucleotide comprising (1) the first codingsequence consisting of a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 17; and, (2) the second coding sequenceconsisting of a polynucleotide consisting of the nucleotide sequence ofSEQ ID NO: 19.

The polynucleotide in a particularly preferred embodiment of the presentinvention includes, for example, a polynucleotide comprising apolynucleotide encoding the fusion protein consisting of the amino acidsequence of SEQ ID NO: 4, 6 or 8, and the like. The polynucleotidecomprising a polynucleotide encoding the fusion protein consisting ofthe amino acid sequence of SEQ ID NO: 4 includes, for example, apolynucleotide comprising a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 3, etc. The polynucleotide comprising apolynucleotide encoding the fusion protein consisting of the amino acidsequence of SEQ ID NO: 6 includes, for example, a polynucleotidecomprising a polynucleotide consisting of the nucleotide sequence of SEQID NO: 5, etc. The polynucleotide comprising a polynucleotide encodingthe fusion protein consisting of the amino acid sequence of SEQ ID NO: 8includes, for example, a polynucleotide comprising a polynucleotideconsisting of the nucleotide sequence of SEQ ID NO: 7, etc.

The polynucleotide of the present invention may further contain apolynucleotide comprising a polynucleotide encoding an additionalpeptide sequence, as in the polynucleotide comprising the polynucleotideconsisting of the nucleotide sequence of SEQ ID NO: 3, 5 or 7. Theadditional peptide sequence includes, for example, at least one peptidesequence selected from the group consisting of a peptide sequence forpromoting translation, a peptide sequence for purification, a secretorysignal peptide sequence, a peptide sequence for expressing the fusionprotein of the present invention as a soluble protein, an epitopesequence capable of recognizing an antibody, etc.

The polynucleotide of the present invention may further contain a linkersequence for a restriction enzyme site, as in the polynucleotidecomprising a polynucleotide consisting of the nucleotide sequence of SEQID NO: 3, 5 or 7.

A polynucleotide comprising a polynucleotide encoding the peptidesequence for promoting translation employed in the art can be used asthe polynucleotide comprising a polynucleotide encoding the peptidesequence for promoting translation. Examples of the peptide sequence forpromoting translation include those described above.

Polynucleotides comprising nucleotide sequences encoding the peptidesequence for purification employed in the art can be used as thepolynucleotide encoding the peptide sequence for purification. Examplesof the peptide sequence for purification include those as describedabove.

Polynucleotides comprising nucleic acids encoding secretory signalpeptides known in the art can be used as the secretory signalpeptide-encoding polynucleotide. Examples of the secretory signalpeptide are those as described above.

The polynucleotide encoding the peptide sequence used to express thefusion protein of the present invention as a soluble protein includes,for example, polypeptides represented by formula (Z)_(n). The amino acidsequences for the polypeptides represented by formula (Z)_(n) and thenucleic acid sequences encoding the same are those as described above.

The linker sequences for restriction enzyme sites employed in the artcan be used as the linker sequences for restriction enzyme sites.

3. Recombinant Vector and Transformant of the Invention

The present invention further provides recombinant vectors andtransformants comprising the polynucleotides of the present inventiondescribed above.

Preparation of Recombinant Vector

The recombinant vector of the invention can be obtained by ligating(inserting) the polynucleotide (DNA) of the invention to (into) anappropriate vector. Specifically, the recombinant vector can be obtainedby digesting the purified polynucleotide (DNA) with a suitablerestriction enzyme, then inserting into a suitable vector at therestriction enzyme site or multicloning site, and ligating to thevector. The vector for inserting the polynucleotide of the invention isnot particularly limited as long as it is replicable in a host. Vectorswhich may be used for this purpose include plasmids, bacteriophages,animal viruses, etc. Examples of plasmids include plasmids from E. coli(e.g., pBR322, pBR325, pUC118, pUC119, etc.), plasmids from Bacillussubtilis (e.g., pUB110, pTP5, etc.), plasmids from yeast (e.g., YEp13,YEp24, YCp50, etc.), and so on. Examples of bacteriophages include kphage, etc. Examples of animal viruses include retroviruses, vacciniaviruses, insect viruses (e.g., baculoviruses), etc. In addition, thepCold I vector, pCold II vector, pCold III vector and pCold IV vector(all are manufactured by Takara-Bio), the PICZ a vector (manufactured byInvitrogen) and the like can also be suitably used.

The polynucleotide of the present invention is generally ligated in anexpressible manner downstream from a promoter in a suitable vector. Whenthe host used for transformation is an animal cell, the promoter ispreferably an SV40-derived promoter, retrovirus promoter,metallothionein promoter, heat shock promoter, cytomegalovirus promoter,SRa promoter, and so on. When the host is a bacterium of the genusEscherichia, Trp promoter, T7 promoter, lac promoter, recA promoter, XPLpromoter, 1 pp promoter, etc. are preferred. When the host is abacterium of the genus Bacillus, SPO1 promoter, SPO2 promoter, penPpromoter, etc. are preferred. When the host is yeast, PHOS promoter, PGKpromoter, GAP promoter, ADH1 promoter, GAL promoter, etc. are preferred.When the host is an insect cell, polyhedrin promoter, P10 promoter, etc.are preferred.

A low-temperature expression-inducible promoter may also be suitablyused. Examples of the low-temperature expression-inducible promoterinclude promoter sequences for cold shock genes, and the like. The coldshock gene includes, for example, Escherichia coli cold shock genes(e.g., cspA, cspB, cspG, cspI, csdA, etc.), Bacillus caldolyticus coldshock genes (e.g., Bc-Csp, etc.), Salmonella enterica cold shock genes(e.g., cspE, etc.), Erwinia carotovora cold shock genes (e.g., cspG,etc.), and the like. Among others, cspA promoter, cspB promoter, cspGpromoter, cspI promoter, csdA promoter and the like can be suitably usedas the low-temperature expression-inducible promoter.

In addition to the foregoing, the recombinant vector of the inventionmay further contain, if desired, an enhancer, a splicing signal, a polyAaddition signal, a ribosome binding sequence (SD sequence), a selectionmarker, etc., and can be provided for use. The selection markerincludes, for example, a dihydrofolate reductase gene, an ampicillinresistance gene, a neomycin resistance gene, etc.

Preparation of Transformant

The thus obtained recombinant vector comprising the polynucleotide ofthe invention (i.e., the polynucleotide encoding the fusion protein ofthe invention) is introduced into an appropriate host, and thetransformant can be prepared. The host is not particularly limited aslong as it is capable of expressing the polynucleotide (DNA) of theinvention. For example, the host may be bacteria of the generaEscherichia, Bacillus, Pseudomonas and Rhizobium, yeast, animal cells orinsect cells, etc. Bacteria of the genus Escherichia include Escherichiacoli, etc. Bacteria of the genus Bacillus include Bacillus subtilis,etc. Bacteria of the genus Pseudomonas include, for example, Pseudomonasputida, etc. Bacteria of the genus Rhizobium include, for example,Rhizobium meliloti, etc. Yeast includes, for example, Saccharomycescerevisiae, Schizosaccharomyces pombe, etc. Animal cells include, forexample, COS cells, CHO cells, etc. Insect cells include, for example,Sf9, Sf21, etc.

The method of transfecting the recombinant vector into the host and themethod of transformation thereby can be performed according to variousgeneral methods. The method for transfecting the recombinant vector intothe host cell includes, for example, the calcium phosphate method(Virology, 52, 456-457 (1973)), the lipofection method (Proc. Natl.Acad. Sci. USA, 84, 7413 (1987)), the electroporation method (EMBO J.,1, 841-845 (1982)), etc. The method for transformation of the bacteriaof the genus Escherichia includes the methods described in, e.g., Proc.Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), etc. Themethod for transformation of the bacteria of the genus Bacillusincludes, for example, the method described in Molecular & GeneralGenetics, 168, 111 (1979), etc. The method for transforming yeastincludes, for example, the method described in Proc. Natl. Acad. Sci.USA, 75, 1929 (1978), etc. The method for transformation of animal cellsincludes, for example, the method described in Virology, 52, 456 (1973),etc. The method for transformation of insect cells includes, forexample, the method described in Bio/Technology, 6, 47-55 (1988), etc.Thus, the transformant transformed with the recombinant vectorcomprising the polynucleotide encoding the protein of the invention(i.e., the polynucleotide of the invention) can be obtained.

Expression Vector and Transformant Comprising Low-TemperatureExpression-Inducible Promoter Sequence

Among others, the expression vector comprising the low-temperatureexpression-inducible promoter sequence is preferred as the expressionvector.

Specifically, the expression vector comprising the low-temperatureexpression-inducible promoter sequence is intended to mean an expressionvector comprising the following promoter sequence and coding sequence:

(1) a low-temperature expression-inducible promoter sequence; and,

(2) a coding sequence comprising the polynucleotide of the invention.

The low-temperature expression-inducible promoter sequence is intendedto mean a promoter sequence which is capable of inducing expression ofthe fusion protein by lowering the temperature from the cultureconditions under which host cells can grow. Examples of thelow-temperature expression-inducible promoter are promoters for geneswhich encode cold shock proteins (cold shock genes). Examples of thecold shock gene promoters include those as described above.

The temperature at which the low-temperature expression-induciblepromoter used in the invention is expression-inducible is generally 30°C. or less, preferably 25° C. or less, more preferably 20° C. or less,and most preferably 15° C. or less. In order to induce the expressionmore efficiently, however, the expression induction is generallyperformed at 5° C. or more, preferably at 10° C. or more, and mostpreferably at approximately 15° C.

In preparing the expression vector of the invention comprising thelow-temperature expression-inducible promoter sequence, the pCold Ivector, pCold II vector, pCold III vector, and pCold IV vector (allmanufactured by Takara-Bio) can be suitably used as the vector forinsertion of the polynucleotide of the invention. The fusion protein ofthe invention can be produced as a soluble protein in the cytoplasmserving as a host when expression is performed in a prokaryotic hostcell using these vectors.

Prokaryotic cells are preferred for the host into which the expressionvector comprising the low-temperature expression-inducible promotersequence is introduced, Escherichia coli being more preferred, the BL21and JM109 strains being particularly preferred. Among others, the BL21strain is most preferred.

Temperatures for incubation at which cell growth is achieved for thetransformant wherein the expression vector comprising thelow-temperature expression-inducible promoter sequence is introduced aregenerally 25 to 40° C. and preferably 30 to 37° C. Temperatures for theexpression induction are generally 4 to 25° C., preferably 10 to 20° C.,more preferably 12 to 18° C., and most preferably 15° C.

4. Production of Fusion Protein of the Invention

The present invention further provides a method for producing the fusionprotein of the invention, which comprises the steps of culturing thetransformant described above and producing the fusion protein of theinvention. The fusion protein of the invention can be produced, forexample, by culturing the transformant described above under conditionswhere the polynucleotide (DNA) encoding the fusion protein of theinvention can be expressed, producing/accumulating the fusion protein ofthe invention and then separating/purifying the protein.

Incubation of Transformant

The transformant of the invention can be incubated in a conventionalmanner used for incubation of a host. By the incubation, the fusionprotein of the invention is produced by the transformant and accumulatedwithin the transformant or in the culture medium.

The medium for culturing the transformant using bacteria of the genusEscherichia or the genus Bacillus as a host may be any of a naturalmedium and a synthetic medium as far as it is a medium which containscarbon sources, nitrogen sources, inorganic salts, etc. necessary forgrowth of the transformant, and in which the transformant canefficiently grow. Examples of carbon sources which can be used arecarbohydrates such as glucose, fructose, sucrose, starch, etc.; organicacids such as acetic acid, propionic acid, etc.; alcohols such asethanol, propanol, and the like. Examples of nitrogen sources which canbe used include ammonia, ammonium salts of inorganic or organic acidssuch as ammonium chloride, ammonium sulfate, ammonium acetate, ammoniumphosphate, etc., and other nitrogen-containing compounds, and furtherinclude peptone, meat extracts, corn steep liquor, and the like.Examples of inorganic salts include monobasic potassium phosphate,dibasic potassium phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,calcium carbonate, etc. If necessary, antibiotics such as ampicillin ortetracycline can be added to the medium during incubation. Where thetransformant transformed by the expression vector using an induciblepromoter as the promoter is cultured, an inducer may also be added tothe medium, if necessary. For example, when the transformant transformedby an expression vector using a Lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside (IPTG), etc. may be added to themedium and indoleacrylic acid (IAA), etc. may be added to the mediumwhen the transformant transformed by an expression vector using a trppromoter is cultured.

When the host is bacteria of the genus Escherichia, incubation isperformed generally at approximately 15 to 43° C. for approximately 3 to24 hours. If necessary, aeration and agitation may be applied. When thehost is bacteria of the genus Bacillus, incubation is performedgenerally at approximately 30 to 40° C. for approximately 6 to 24 hours.If necessary, aeration and agitation may be applied.

Media for incubation of the transformant when the host is yeast includeBurkholder's minimal medium (Proc. Natl. Acad. Sci. USA, 77, 4505(1980)) and an SD medium containing 0.5% (w/v) Casamino acids (Proc.Natl. Acad. Sci. USA, 81, 5330 (1984)). Preferably, the pH of the mediumis adjusted to approximately 5 to 8. Incubation is performed generallyat approximately 20 to 35° C. for approximately 24 to 72 hours. Ifnecessary, aeration and agitation may be applied.

Media for culturing the transformant when the host is an animal cellinclude MEM medium supplemented with approximately 5 to 20% (v/v) fetalcalf serum (Science, 122, 501 (1952)), DMEM medium (Virology, 8, 396(1959)), etc. Preferably, the pH of the medium is adjusted toapproximately 6 to 8. Incubation is performed generally at approximately30 to 40° C. for approximately 15 to 60 hours. If necessary, aerationand agitation may be applied.

Media for culturing the transformant when the host is an insect cellinclude Grace's insect medium (Nature, 195, 788 (1962)) to whichadditives such as 10% (v/v) immobilized bovine serum are suitably added.Preferably, the pH of the medium is adjusted to approximately 6.2 to6.4. Incubation is performed generally at approximately 27° C. forapproximately 3 to 5 hours. If necessary, aeration and agitation may beapplied.

Temperatures for incubation at which the transformant transformed by theexpression vector comprising the low-temperature expression-induciblepromoter sequence and temperatures for expression induction are asdescribed above.

Separation/Purification of Fusion Protein of the Invention

The fusion protein of the present invention can be obtained byseparating/purifying the fusion protein of the present invention fromthe culture described above. As used herein, the culture is intended tomean any one of a culture broth, cultured cells or cultured bacteria anda cell lysate of the cultured cells or cultured bacteria. The fusionprotein of the present invention can be separated/purified in aconventional manner.

Specifically, when the fusion protein of the present inventionaccumulates in the cultured bacteria or cultured cells, after completionof the incubation, the bacteria or cells are disrupted in a conventionalmanner (e.g., ultrasonication, lysozyme, freezing and thawing, etc,) andthen a crude extract of the fusion protein of the invention can beobtained in a conventional manner (e.g., centrifugation, filtration,etc.). When the fusion protein of the invention accumulates in theperiplasmic space, after completion of the incubation, the extractcontaining the fusion protein of the invention can be obtained in aconventional manner (e.g., the osmotic shock method, etc.). When thefusion protein of the invention accumulates in the culture broth, aftercompletion of the incubation, the culture supernatant containing thefusion protein of the invention can be obtained by separating thebacteria or cells and the culture supernatant in a conventional manner(e.g., centrifugation, filtration, etc.).

The fusion protein of the invention contained in the extract or culturesupernatant thus obtained can be purified by conventional methods ofseparation and purification. Examples of these separation andpurification methods which may be used include ammonium sulfateprecipitation, gel filtration chromatography, ion-exchangechromatography, affinity chromatography, reversed-phase high-performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or in asuitable combination thereof. If the fusion protein of the inventioncontains the peptide sequence for purification described above, it ispreferred to perform the purification using the same. Specifically, whenthe fusion protein of the invention contains a histidine tag sequence,nickel chelate affinity chromatography may be used; when the fusionprotein of the invention contains the binding domain of S-transferase toglutathione, affinity chromatography with a glutathione-binding gel maybe used; when the fusion protein of the invention contains the aminoacid sequence of Protein A, antibody affinity chromatography may beused.

5. Complex of the Invention

The fusion protein of the invention (hereinafter sometimes referred toas “the luciferase of the invention”) can bind to other useful compound(e.g., a fluorescent substance, a ligand specific to an analyte to bedetected, etc.) to form a complex.

The complex of the present invention comprises the luciferase of theinvention and other useful compound bound to the second region of theluciferase via the thiol group of the cysteine residues. In someembodiments of the present invention, the complex comprises theluciferase of the invention bound to a fluorescent substance or a ligandspecific to an analyte. In the complex in some embodiments of thepresent invention, the binding ratio of the thiol group of theluciferase to the fluorescent substance or ligand is 1:1 or a ratioclose thereto.

Among the other useful compounds, the fluorescent substance includes anorganic compound such as Hoechist3342, Indo-1, DAP1, etc.; a fluorescentprotein such as a green fluorescent protein (GFP), a blue fluorescentprotein (BFP), a mutant GFP fluorescent protein, phycobilin, etc.

The ligand specific to an analyte may be any one of a substance directlybinding to the analyte and a substance indirectly binding to theanalyte. The ligand includes, for example, biotin, biotin-conjugatedproteins, enzymes, substrates, antibodies, antigens, nucleic acids,polysaccharides, receptors and compounds capable of binding to thesesubstances.

Among them, the biotin-conjugated proteins include avidin, streptavidin,mutant avidin (NeutrAvidin), etc. These biotin-conjugated proteins mayalso be obtained from commercially available ones. Also, thebiotin-conjugated proteins which are commercially available may beprepared so as to be modifiable.

Antigens against a variety of substances (e.g., trace components ofhuman, animal or plant origin in vivo such as tumor markers, hormones,etc., environmental trace pollutants, etc.) and antibodies against theseantigens have been commercially marketed so far. Antibodies that ananalyte to be determined is an antigen can be obtained appropriatelydepending upon necessity and provided for use.

As markers which increase in blood serum or urine accompanied by tumorgrowth, inter alia, there are known so far markers which specificallyincrease in each organ accompanied by tumor formation, such as fetalantigen, CA19-9, sialyl Lex-i antigen, sialyl Tn antigen, thymidinekinase activity, tissue polypeptide antigen, basic fetoprotein,immunosuppressive acidic protein, CA72-4, CAl25, DUPAN-2, SPan-1,elastase 1, BCA-225, CA15-3, SCC antigen, cytokeratin 19 fragment,prostate specific antigen, γ-seminoprotein, prostate acidic phosphatase,α-fetoprotein, AFP lectin fraction, PIVKA-II, neuron specific enolase,NCC-ST-439, CA130, type I collagen-C-telopeptide, and the like. Theseantigens are commercially available and may be suitably used as thestandard substance in monitoring these markers in blood serum or urine.In addition, antibodies from various classes or subclasses against theseantigens are commercially available and can be appropriately used.

The nucleic acids may be optional complementary DNAs and RNAs, andinclude, for example, DNAs and RNAs having nucleotide sequences whichcan be used for pathogenic gene detection, gene diagnosis, etc. Thesenucleic acids can be chemically synthesized appropriately in aconventional manner.

Taking into account the molecular size of the luciferase of theinvention and steric hindrance with the other useful compound, the otheruseful compound is bound to the luciferase of the invention directly orvia a linker or spacer, though it varies depending upon physicochemicalproperties or the like.

The linker or spacer used in the present invention is not particularlylimited as far as it is capable of specifically reacting with the —SHgroups, but preferably has a length of 20 angstroms or more. Variousreagents for modifying —SH groups which can be used as the linker orspacer are commercially available and can be appropriately utilized.

The crosslinking reagents with a functional group reactive with thethiol group (also called as “sulfhydryl group”) of cysteines are notparticularly limited. Specific examples areN-(4-[p-azidosalicylamido]butyl]-3′-(2′-pyridyldithio)propionamide(APDP), 1,4-di-(3′-[2′-pyridyldithio]propionamide)butane,1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl3-(bromoacetamide)propionate (SBAP), N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),N-[α-maleimidoacetoacetoxy]succinimide ester (AMAS), succinimidyl4-(N-maleimidomethyl)cyclohexane (SMCC), sulfonated derivatives of SMCC(sulfo-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),sulfonated derivatives of MBS (sulfo-MBS), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), sulfonated derivatives of SMPB(sulfo-SMPB), succinimidyl 6-(N-maleimido-n-hexanoate), succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-hydroxysuccinimidyl bromoacetate,bis-(maleimido)methyl ester, bis-maleimidohexane (BMH), etc. Among thereagents above, crosslinking reagents having a maleimide group as thefunctional group capable of reacting with the sulfhydryl group areparticularly preferred in the present invention.

The reaction of binding the luciferase of the invention to the otheruseful compound can be carried out by known methods in the art, e.g., bythe method described in Hermanson G. T., Bioconjugate Techniques, 2ndEdition (2008), Elsevier Inc., etc. More specifically, when the ligandspecific to an analyte is used, it is desired to perform the reaction at30° C. or less and preferably 25° C. or less, in pH 6 to 8 andpreferably pH 7 to 7.5. In the case of the fluorescent substance, it isdesired to perform the reaction at 25° C. or less and preferably 4° C.or less, in pH 6 to 8, and preferably pH 7 to 7.5.

Especially when the biotin-conjugated protein is used, it is desired toperform the reaction at 25° C. or less and preferably 4° C. or less, inpH 6 to 8 and preferably pH 7 to 7.5. In the case of the antibody, it isdesired to perform the reaction at 25° C. or less and preferably 4° C.or less, in pH 6 to 8 and preferably pH 7 to 7.5. In the case of thenucleic acid, it is desired to perform the reaction at 25° C. or lessand preferably 4° C. or less, in pH 6 to 8 and preferably pH 7 to 7.5.

6. Use of Fusion Protein and Complex of the Invention Use as DetectionMarker by Luminescence

The fusion protein of the invention or the complex of the invention canbe utilized as a detection marker which emits luminescence in thepresence of a luciferin. The detection marker of the present inventioncan be utilized for detection of the target substance in, e.g., animmunoassay, a hybridization assay, etc. When the complex of theinvention is used as a detection marker, the other useful compound inthe complex of the invention is a ligand specific to the analyte to bedetected.

When the complex of the invention is used in an immunoassay, the ligandto the complex of the invention includes, for example, a primaryantibody which specifically recognizes the target substance. The primaryantibody in the complex of the invention specifically binds to ananalyte (antigen) present in a sample. Thus, the site or amount of theanalyte in the sample can be detected by monitoring luminescence of thefusion protein in the complex of the invention.

In order to enhance the detection sensitivity, there is also well knowna method of using a secondary antibody which specifically recognizes theprimary antibody. In this case, the ligand in the complex of theinvention is, e.g., a secondary antibody.

Alternatively, a biotin-conjugated secondary antibody formed byconjugating biotin to a secondary antibody can be reacted with avidin orstreptavidin conjugated with the fusion protein of the invention. Inthis case, the ligand in the complex of the invention is avidin orstreptavidin.

Furthermore, the property of binding 1 molecule of avidin or 1 moleculeof streptavidin to 4 molecules of biotin can be utilized. Morespecifically, a biotinylated secondary antibody is reacted with avidinor streptavidin, which is in turn reacted with biotin conjugated to thefusion protein of the invention. In this case, the ligand in the complexof the invention is biotin.

When a receptor is detected using the complex of the invention, a signalpeptide (hormones such as insulin, cytokines, TNF, Fas ligands, etc.)can be used as a ligand in the complex of the invention. On the otherhand, when the signal peptide is detected, a protein which constitutesthe receptor can be used as a ligand in the complex of the invention.That is, in detecting a receptor to which a drug is bound, the drug canbe used as the ligand in the complex of the invention, and in detectinga drug bound to a receptor, the receptor can be used as a ligand in thecomplex of the invention.

When an enzyme is detected using the complex of the invention, asubstrate to the enzyme can be used as a ligand in the complex of theinvention. On the other hand, when a substrate to the enzyme isdetected, the enzyme can be used as a ligand in the complex of theinvention.

When the complex of the invention is utilized in a hybridization assay,in order to detect another nucleic acid specifically binding to acertain nucleic acid, a nucleic acid complementary to the nucleic acidto be detected can be used as a ligand in the complex of the invention.

When another substance specifically binding to a polysaccharide isdetected using the complex of the invention, the polysaccharide can beused as a ligand in the complex of the invention.

In addition to the foregoing, lectin capable of specifically binding toa blood coagulation factor or a DNA-binding protein such as atranscription factor, etc. can also be used as a ligand in the complexof the invention.

The complex of the invention can be bound directly or indirectly to thetarget substance via a ligand, as described above, and can thus beutilized as a detection marker which emits light in the presence of aluciferin. Detection of the target substance using such a detectionmarker can be performed in a conventional manner.

Furthermore, the fusion protein of the invention can be expressed, e.g.,as a fusion protein with a target protein, and introduced into cells bymeans of the microinjection method, etc., and the resulting product canbe used to determine distribution of the target protein described above.The distribution of such a target protein or the like can be determinedby using detection methods such as luminescence imaging. In addition tothe introduction into cells by means of the microinjection method or thelike, the fusion protein of the invention can be expressed in cells andprovided for use.

Use as Reporter Protein

The fusion protein of the invention may also be used as a reporterprotein to assay the transcription activity of promoters, etc. Thepolynucleotide encoding the fusion protein of the invention (i.e., thepolynucleotide of the invention) is fused to a target promoter or someother expression control sequence (e.g., an enhancer, etc.) to constructa vector. By introducing the vector into a host cell and detecting theluminescence from the fusion protein of the invention in the presence ofa luciferin, the activity of the target promoter or some otherexpression control sequence can be assayed.

The polynucleotide of the invention can be used as a reporter gene insuch a manner as described above.

Material for Amusement Supplies

The fusion protein of the invention has the activity of catalyzing thereaction where luciferin is oxidized with oxygen molecules to formoxyluciferin in its excited state. The oxyluciferin in the excited stateemits visible light to decay to the ground state. Accordingly, thefusion protein, etc. of the invention can be used preferably as aluminescent material for amusement supplies. Examples of such amusementsupplies are luminescent soap bubbles, luminescent ice bars, luminescentcandies, luminescent color paints, etc. These amusement supplies can beprepared in a conventional manner.

Bioluminescence Resonance Energy Transfer (BRET) Method

By utilizing the principle of interaction between molecules by thebioluminescence resonance energy transfer (BRET) method, the fusionprotein of the invention or the complex of the invention is availablefor analytical methods such as analysis of physiological functions,assay of enzyme activities, etc.

For instance, when the fusion protein of the invention or the complex ofthe invention (sometimes referred to as “the luciferase of theinvention”) is used as a donor and the fluorescent substance (e.g., anorganic compound, a fluorescent protein, etc.) is used as an acceptor,the interactions between the donor and acceptor above can be detected bycausing bioluminescence resonance energy transfer (BRET) between them.

In an embodiment of the present invention, the organic compound used asan acceptor includes Hoechist3342, Indo-1, DAP1, etc. In anotherembodiment of the present invention, the fluorescent protein used as anacceptor includes a green fluorescent protein (GFP), a blue fluorescentprotein (BFP), a mutant GFP fluorescent protein, phycobilin, etc.

In a preferred embodiment of the present invention, the physiologicalfunctions to be analyzed include an orphan receptor (especially, a Gprotein-coupled receptor), apoptosis, transcription regulation by geneexpression, etc. Further in a preferred embodiment of the presentinvention, the enzyme to be analyzed is protease, esterase, kinase, orthe like.

Analysis of the physiological functions by the BRET method can beperformed by known methods, for example, by modifications of the methodsdescribed in Biochem. J. 2005, 385, 625-637, Expert Opin. Ther Tarets,2007 11: 541-556, etc. Measurement of enzyme activities may also beperformed by known methods, for example, by modifications of the methodsdescribed in Nature Methods 2006, 3:165-174, Biotechnol. J. 2008,3:311-324, etc.

In the complex of some embodiments of the present invention, the fusionprotein of the invention is conjugated with the fluorescent substance asthe other useful compound. The fluorescent substance includes thosedescribed above. When the fusion protein is used as a donor in thecomplex of the invention and the fluorescent substance is used as anacceptor in the same complex, BRET can also be caused between them.

7. Kit of the Invention

The present invention further provides a kit comprising any one selectedfrom the fusion protein of the invention, the polynucleotide of theinvention, the recombinant vector of the invention, the transformant ofthe invention and the complex of the invention. The kit of the presentinvention may further contain a luciferin (e.g., a coelenterazineanalogue). The kit of the present invention can be prepared withconventional materials by conventional methods. The kit of the presentinvention may further contain sample tubes, plates, instructions for thekit user, solutions, buffers, reagents, and samples suitable forstandardization or control samples. The kit of the present invention mayfurther contain salts including halide ions.

The kit of the present invention can be used for the aforesaidmeasurement using a reporter protein or a reporter gene, the analysis ofphysiological functions or measurement of enzyme activities by the BRETmethod.

8. Method for Luminescence Reaction Catalytic Ability for LuminescenceActivity

The fusion protein of the invention or the complex of the invention(hereinafter sometimes referred to as “the fusion protein, etc. of theinvention”) has the ability of catalyzing the reaction in which aluciferin (e.g., a coelenterazine analogue) is oxidized with oxygenmolecules to form an oxyluciferin in its excited state. The oxyluciferinin the excited state emits light on returning to the ground state. Thatis, the fusion protein, etc. of the invention catalyzes the luminescencereaction in which a luciferin (e.g., a coelenterazine analogue) servesas a substrate to cause luminescence. This activity is sometimesreferred to as “the catalytic ability for the luminescence activity.”

Luminescence Reaction

The luminescence reaction using the fusion protein, etc. of theinvention in which a luciferin (e.g., a coelenterazine analogue) servesas a substrate can be performed by contacting the fusion protein, etc.of the invention with the luciferin. The reaction can be carried outunder conditions conventionally used for the luminescence reaction usingGaussia luciferase or under those modified therefrom (see, e.g., WO99/49019, J. Biol. Chem. 279, 3212-3217 (2004) and the documents citedtherein, etc.).

Specifically, solvents for the reaction which are employed are, forexample, a buffer solution such as Tris-HCl buffer, sodium phosphatebuffer, etc., water, and the like.

Temperatures for the reaction are generally at approximately 4° C. to40° C. and preferably approximately 4° C. to 25° C.

In the reaction solution, pH is generally approximately 5 to 10,preferably approximately 6 to 9, more preferably approximately 7 to 8and most preferably approximately 7.5

Coelenterazine analogues are preferred as the luciferin, with particularpreference being coelenterazine.

The luciferin may also be added to the reaction system in the form of asolution in a polar solvent such as dimethylformamide,dimethylsulfoxide, etc., or in an alcohol such as methanol, ethanol,butanol, etc.

Activation of Luminescence Activity

The luminescence activity of the fusion protein, etc. of the inventionwhere the luciferin (e.g., coelenterazine analogue) serves as asubstrate is activated by halide ions.

Examples of the halide ions are fluorine ions, chlorine ions, bromineions and iodine ions; preferred are chlorine ions, bromine ions andiodine ions.

The concentration of the halide ions is generally approximately 10 μM to100 mM, preferably approximately 100 μM to 50 mM and particularlypreferably approximately 1 mM to 20 mM.

To add the halide ions to the reaction system, there is a method whichcomprises adding them in a salt form. The salts used are alkali metalsalts such as sodium salts, potassium salts, etc.; alkaline earth metalsalts such as calcium salts, magnesium salts, barium salts, etc. Morespecific examples are NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI, CaF₂,CaCl₂, CaBr₂, CaI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, etc.

Unless otherwise indicated with respect to the embodiments and workingexamples, the methods described in standard sets of protocols such as J.Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, alaboratory manual (3rd edition), Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D.Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols inMolecular Biology, John Wiley & Sons Ltd., or modifications orvariations thereof are used. When commercially available reagent kits ormeasuring apparatuses are used, protocols attached to them are usedunless otherwise indicated.

Regardless of their purposes, all of the documents and publicationsdescribed in the specification are incorporated herein by reference,each in its respective entirety.

The objects, characteristics and advantages of the present invention aswell as the idea thereof are apparent to those skilled in the art fromthe descriptions given herein. Based on the description given herein,those skilled in the art can easily work the present invention. It is tobe understood that the best mode for carrying out the invention,specific working examples, etc. are to be taken as preferred embodimentsof the present invention. These descriptions are only for illustrativeand explanatory purposes and are not intended to restrict the inventionthereto. It is further apparent to those skilled in the art that variousmodifications may be made based on the descriptions given herein withinthe intent and scope of the present invention disclosed herein.

EXAMPLES

Hereinafter, the present invention will be described with reference toEXAMPLES below but is not deemed to limit the invention thereto.

Example 1 Construction of Novel Expression Vector Having Hinge Sequencein Yeast

A novel expression vector having a hinge sequence was constructed asfollows. First, the following linkers, pPICZα Linker-F and pPICZαLinker-R having chemically synthesized multicloning sequences wereinserted into the restriction enzyme XhoI/S all sites in pPICZα A(Invitrogen) to construct the novel vector pPICZα-Linker.

pPICZα Linker-F (SEQ ID NO: 9) (5′TC GAA AAA AGA GAG GCT GAA GCT GGT ACC GAA TTCCTG CAG CTC GAG TCT AGA G 3′) pPICZα Linker-R (SEQ ID NO: 10) (5′TC GAC TCT AGA CTC GAG CTG CAG GAA TTC GGT ACCAGC TTC AGC CTC TCT TTT T 3′)

Next, the following linkers: hinge Linker-F and hinge Linker-Rcontaining a chemically synthesized multicloning sequence were insertedinto the restriction enzyme PstI/SalI sites of the pPICZα-Linker vectorto construct the novel expression vector pPICZα-hgLinker having a hingesequence, which is shown in FIG. 1.

hinge Linker-F (5′ G AGC TTA TCC ACC CCG CCG ACC CCG TCC CCG TCC ACC CCGCCG TGC CTC GAG TCT AGA G 3′; the new cysteine residue underlined) (SEQID NO: 11)

hinge Linker-R (5′ TC GAC TCT AGA CTC GAG GCA CGG CGG GGT GGA CGG GGACGG GGT CGG CGG GGT GGA TAA GCT CTG CA 3′; the newly introduced cysteineresidue underlined) (SEQ ID NO: 12)

The insert DNA was confirmed by nucleotide sequencing on a DNA Sequencer(manufactured by ABI). The DNA sequence of the expression vectorpPICZα-hgLinker is shown by SEQ ID NO: 1. The amino acid sequence of aprotein encoded by the DNA sequence of the expression vectorpPICZα-hgLinker is shown by SEQ ID NO: 2.

Example 2 Construction and Gaussia Luciferase Expression Vector in Yeast

The Gaussia luciferase expression vector with a hinge sequence for thepurpose of expression in yeast was constructed as follows. UsingpcDNA3-hGL as a template, PCR was performed (cycle conditions of 25cycles: 1 min/94° C., 1 min/50° C. and 1 min/72° C.) using the two PCRprimers shown below to amplify the desired DNA region.

Primer: GL6-N/EcoRI (SEQ ID NO: 13) (5′gcc GAA TTC AAG CCC ACC GAG AAC AAC GAA 3′) Primer: GL26C-TAA/PstI(SEQ ID NO: 14) (5′ ggc CTG CAG GTC ACC ACC GGC CCC CTT GAT 3′)

The resulting fragment was purified by a PCR Purification Kit(manufactured by Qiagen). After digestion with restriction enzymesEcoRI/PstI in a conventional manner, the fragment was ligated to thepPICZα-hgLinker constructed in EXAMPLE 1 at the restriction enzymeEcoRI/PstI sites. The yeast expression vector pPICZα-hgGL-H shown inFIG. 2 was thus constructed.

The insert DNA was confirmed by nucleotide sequencing on a DNA Sequencer(manufactured by ABI). The DNA sequence encoding the hg-Gaussialuciferase fusion protein inserted into the expression vectorpPICZα-hgGL-His shown by SEQ ID NO: 3. The amino acid sequence of thehg-Gaussia luciferase fusion protein inserted into the expression vectorpPICZα-hgGL-His shown by SEQ ID NO: 4.

For expression in yeast, the expression vector pPICZα-hgGL-H wasintroduced into yeast X33 strain (manufactured by Invitrogen) on aconventional electroporation apparatus (manufactured by Bio-Rad) andcultured at 30° C. for 2 days on agar of YM medium (manufactured byDifco) containing Zeocin (100 mg/ml) to obtain the transformant. Thetransformant was cultured at 30° C. for 18 hours in liquid YM medium(manufactured by Difco) containing 10 ml of Zeocin (100 mg/ml). Aftercentrifugation, the bacterial cells were isolated. Coelenterazine(Chisso Corporation, hereinafter the same) as a luminescence substratewas added to the supernatant to confirm the luminescence activity.

Example 3 Construction of Gaussia Luciferase Expression Vector in E.coli

The Gaussia luciferase expression vector with a hinge sequence for thepurpose of expression in E. coli was constructed as follows. Theexpression vector pPICZα-hgGL-H constructed in EXAMPLE 2 was digestedwith restriction enzymes Asp718I/XbaI in a conventional manner. Theresulting fragment was inserted into pColdII (manufactured byTakara-Bio) at the restriction enzyme Asp718I/XbaI sites to constructthe expression vector pCold-hgGL shown in FIG. 3.

The insert DNA was confirmed by nucleotide sequencing on a DNA Sequencer(manufactured by ABI). The DNA sequence encoding the hg-Gaussialuciferase fusion protein, which was inserted into the expression vectorpCold-hgGL, is shown by SEQ ID NO: 5. The amino acid sequence of thehg-Gaussia luciferase fusion protein, which was inserted into theexpression vector pCold-hgGL, is shown by SEQ ID NO: 6.

Example 4 Construction of Gaussia Luciferase Expression Vector Having aHinge Sequence and an Avidin Tag Sequence in E. coli

Gaussia luciferase expression vector having a hinge sequence and anavidin tag sequence was constructed as follows. The following linkersAvitag-Xho/Xba-F and AviTag-Xho/Xba-R having a chemically synthesizedmulticloning site were inserted into the XhoI/XbaI sites, which are therestriction enzyme sites for the expression vector pCold-hgGLconstructed in EXAMPLE 3. Thus, novel expression vector pCold-hgA-GLshown in FIG. 4 was constructed.

Avitag-Xho/Xba-F (SEQ ID NO: 15) (5′TC GAG GGT CTG AAC GAC ATC TTC GAA GCT CAG AAA ATC GAA TGG CAC GAA T 3′)AviTag-Xho/Xba-R (SEQ ID NO: 16) (5′CT AGA TTC GTG CCA TTC GAT TTT CTG AGC CTC GAA GAT GTC GTT CAG ACC C 3′)

The insert DNA was confirmed by nucleotide sequencing on a DNA Sequencer(manufactured by ABI). The DNA sequence encoding the hg-Gaussialuciferase fusion protein, which was inserted into the expression vectorpCold-hgA-GL, is shown by SEQ ID NO: 7. The amino acid sequence of thehg-Gaussia luciferase fusion protein, which was inserted into theexpression vector pCold-hgA-GL, is shown by SEQ ID NO: 8.

Example 5 Expression and Purification of Recombinant Gaussia LuciferaseHaving the Hinge Sequence from E. coli

The recombinant hg-Gaussia luciferase having the hinge sequence(hg-Gaussia luciferase) was expressed in E. coli using the expressionvector pCold-hgGL. The recombinant hg-Gaussia luciferase was purified bynickel-chelate column chromatography and then hydrophobic columnchromatography.

1) Expression of Recombinant hg-Gaussia Luciferase Having the HingeSequence in E. coli

To express the recombinant hg-Gaussia luciferase in E. coli, the Gaussialuciferase gene expression vector pCold-hgGL constructed in EXAMPLE 3was used. The expression vector was transformed into the E. coli BL21strain in a conventional manner. The transformant obtained wasinoculated in 10 ml of LB liquid medium (10 g of bactotryptone, 5 g ofyeast extract and 5 g of sodium chloride per 1 liter of water, pH 7.2)containing ampicillin (50 μg/ml) and incubated at 37° C. for 18 hours.Subsequently, the culture broth was added to 400 ml×5 (2000 ml in total)of a fresh LB liquid medium. After culturing at 37° C. for 5 hours, themixture was cooled on an ice water.Isopropyl-β-D(−)-thiogalactopyranoside (IPTG, manufactured by Wako PureChemical Industry) was added to the culture to a final concentration of0.1 mM, followed by incubation at 15° C. for 17 hours. The cells wererecovered by centrifugation (5,000 rpm, 5 mins.) and provided for use asthe starting material for protein extraction.

2) Extraction of hg-Gaussia Luciferase from Supernatant of CulturedCells and Nickel-Chelate Gel Column Chromatography

The cultured cells collected were suspended in 200 ml of 50 mM Tris-HCl(pH 7.6) and disrupted by ultrasonication (manufactured by Branson,Sonifier Model Cycle 250) 3 times each for 3 minutes under ice cooling.The cell lysate was centrifuged at 10,000 rpm (12,000×g) at 4° C. for 20minutes. The resultant soluble fractions were applied on anickel-chelate column (Amersham Bioscience, column size: diameter 2.5×6cm) equilibrated with 50 mM Tris-HCl (pH 7.6) to adsorb hg-Gaussialuciferase. After washing with 300 ml of 50 mM Tris-HCl (pH 7.6),hg-Gaussia luciferase was eluted with 0.1M imidazole (manufactured byWako Pure Chemical Industry). From 800 ml of the cultured cells, 50.4 mgof hgGL was obtained. As a result of SDS-PAGE analysis, the purity wasestimated to be over 95%

3) Purification of hg-Gaussia Luciferase by Hydrophobic ColumnChromatography

After (NH₄)₂SO₄ was added to 63 ml of the fraction (50.4 mg as theprotein) eluted from the nickel-chelate gel column to a finalconcentration of 1.2 M, the mixture was centrifuged at 10,000 rpm(12,000×g) at 4° C. for 20 minutes. The resultant soluble fractions wereapplied to a butyl-Sepharose column (Amersham Bioscience, column size:diameter 2.5×5.5 cm) equilibrated with 10 mM Tris-HCl (pH 7.6) to adsorbhg-Gaussia luciferase thereto. After washing with 110 ml of 10 mMTris-HCl (pH 7.6) containing 1.2 M (NH₄)₂SO₄ and 2 mM EDTA, hg-Gaussialuciferase was eluted with 10 mM Tris-HCl (pH 7.6) containing 0.4M(NH₄)₂SO₄ and 2 mM EDTA, and 6.5 mg of hg-Gaussia luciferase wasobtained. As a result of SDS-PAGE analysis, the purity was estimated tobe over 95%.

4) Quantification of Protein Concentration

Protein concentration was determined by the method of Bradford using acommercially available kit (manufactured by Bio-Rad) and bovine serumalbumin (manufactured by Pierce) as a standard.

For the fraction in each purification step, SDS-PAGE analysis wasperformed on a 12% polyacrylamide gel under reducing conditions. Asshown in FIG. 5, the results revealed that a single band correspondingto the protein of 22 kDa molecular weight was detected and the puritywas over 95%. The activity recovery of hg-Gaussia luciferase from thesoluble fraction obtained from 2000 ml of cultured cells was 38.4% andthe yield was 6.5 mg.

5) Determination of Luminescence Activity

After 1 μl of a Gaussia luciferase solution was added to 0.1 ml of PBS(manufactured by Sigma; 0.137M sodium chloride and 0.0027M potassiumchloride, pH 7.4) supplemented with 0.01% Tween 20 and 10 mM EDTA(manufactured by Wako Pure Chemical Industry) (hereinafter referred toas PBST-E), which contained 0.5 μg of substrate coelenterazine, theluminescence activity of Gaussia luciferase was measured for 10 secondsusing a luminescence luminometer: Luminescencer-PSN AB2200 (manufacturedby Atto). The luminescence activity is shown in terms of the maximumintensity (Imax).

TABLE 1 Purification of hg-Gaussia Luciferase Total Total protein TotalSpecific amount concentration activity activity Recovery rate (%) Step(ml) (mg) (×10⁹ rlu) (×10⁹/mg) Protein Activity Crude extract from 200760 178.4 0.24 100 100 the soluble fractions Nickel-chelate gel 63 50.4111.5 2.21 6.6 62.5 Butyl-Sepharose gel 39 6.5 68.5 10.6 0.9 38.4

Example 6 Expression and Purification of Recombinant Gaussia LuciferaseHaving the Avidin Tag Sequence from E. coli

The recombinant Gaussia luciferase having the avidin tag sequence(hgA-Gaussia luciferase) was expressed in E. coli using the expressionvector pCold-hgA-GL. The hgA-Gaussia luciferase was purified bynickel-chelate column chromatography and then hydrophobicchromatography.

1) Expression of Recombinant hgA-Gaussia Luciferase Having the AvidinTag Sequence in E. Coli

The Gaussia luciferase gene expression vector pCold-hgA-GL constructedin EXAMPLE 4 was used to express the recombinant hgA-Gaussia luciferasein E. coli. The expression vector was transformed into the E. coli BL21strain in a conventional manner. The transformant obtained wasinoculated into 10 ml×2 tubes of LB liquid medium (10 g ofbactotryptone, 5 g of yeast extract and 5 g of sodium chloride per 1liter of water, pH 7.2) containing ampicillin (50 μg/ml), followed byincubation at 37° C. for 18 hours. Next, the culture broth was added to400 ml×2 tubes of fresh LB liquid medium (800 ml in total). Afterincubation at 37° C. for 3 hours, the culture broth was cooled in icewater. Isopropyl-β-D(−)-thiogalactopyranoside (IPTG, manufactured byWako Pure Chemical Industry) was added to the culture broth to a finalconcentration of 0.1 mM, followed by incubation at 15° C. for 19 hours.After the incubation, the cells were recovered by centrifugation (5,000rpm, 5 mins.) and provided for use as the starting material for proteinextraction.

2) Extraction of hgA-Gaussia Luciferase from Supernatant of CulturedCells and Nickel-Chelate Column Chromatography

The cultured cells collected were suspended in 80 ml of 50 mM Tris-HCl(pH 7.6) and disrupted by ultrasonication (manufactured by Branson,Sonifier Model Cycle 250) 4 times each for 3 minutes under ice cooling.The cell lysate was centrifuged at 10,000 rpm (12,000×g) at 4° C. for 20minutes. The resultant soluble fractions were applied onto anickel-chelate column (Amersham Bioscience, column size: diameter 2.5×6cm) equilibrated with 50 mM Tris-HCl (pH 7.6) to adsorb hgA-Gaussialuciferase. After washing with 50 mM Tris-HCl (pH 7.6), hgA-Gaussialuciferase was eluted with 0.1M imidazole (manufactured by Wako PureChemical Industry). From 800 ml of the cultured cells, 19.9 mg ofhgA-Gaussia luciferase was obtained.

3) Purification of hgA-Gaussia Luciferase by Hydrophobic ColumnChromatography

After (NH₄)₂SO₄ was added to 39 ml of the fraction (19.9 mg as theprotein) eluted from the nickel-chelate column to a final concentrationof 1.2 M, the mixture was centrifuged at 10,000 rpm (12,000×g) at 4° C.for 20 minutes. The resultant soluble fractions were applied to abutyl-Sepharose column (Amersham Bioscience, column size: diameter2.5×5.5 cm) equilibrated with 10 mM Tris-HCl (pH 7.6) to adsorbhgA-Gaussia luciferase. After washing with 10 mM Tris-HCl (pH 7.6)containing 1.2 M (NH₄)₂SO₄ and 2 mM EDTA, hgA-Gaussia luciferase waseluted with 10 mM Tris-HCl (pH 7.6) containing 0.4 M (NH₄)₂SO₄ and 2 mMEDTA to give 0.92 mg of hgA-Gaussia luciferase. As a result of SDS-PAGEanalysis, the purity was estimated to be 95% or more.

4) Quantification of Protein Concentration

Protein concentration was determined by the method of Bradford using acommercially available kit (manufactured by Bio-Rad) and bovine serumalbumin (manufactured by Pierce) as a standard.

SDS-PAGE analysis of the fraction in each purification step wasperformed on a 12% polyacrylamide gel under reducing conditions. Asshown in FIG. 6, the results revealed that a single band correspondingto the protein of 29 kDa molecular weight was detected and the puritywas over 95%. The activity recovery rate of hgA-Gaussia luciferase from800 ml of the cultured cells was 15.8% and the yield was 0.92 mg.

5) Determination of Luminescence Activity

After 1 μl of a Gaussia luciferase solution was added to 0.1 ml of PBS(manufactured by Sigma; 0.137 M sodium chloride and 0.0027 M potassiumchloride, pH 7.4) supplemented with 0.01% Tween 20 and 10 mM EDTA(manufactured by Wako Pure Chemical Industry) (hereinafter referred toas PBST-E), which contained 0.5 μg of substrate coelenterazine, theluminescence activity was measured for 10 seconds using an apparatus formeasuring luminescence: Luminescencer-PSN AB2200 (manufactured by Atto).The luminescence activity is shown in terms of the maximum intensity(Imax).

TABLE 2 Purification of hgA-Gaussia Luciferase Total Total protein TotalSpecific amount concentration activity activity Recovery rate (%) Step(ml) (mg) (×10⁹ rlu) (×10⁹/mg) Protein Activity Crude extract from 80224 55.6 0.25 100 100 the soluble fractions Nickel-chelate gel 39 19.963.4 3.19 8.9 114 Butyl-Sepharose gel 40 0.92 8.8 9.57 0.09 15.8

Example 7 Preparation of Biotinylated hg-Gaussia Luciferase byMaleimide-Activated Biotin

To 500 μl of a PBS solution supplemented with 1 mM EDTA (hereinafterreferred to as PBS-E), 4.4 μl (4.4 nmol) of maleimide-activated biotin(manufactured by Pierce, EZ-Link PEO-Maleimide-Activated Biotin; spacerlength: 29.1 angstroms) adjusted to 1 mM with PBS-E, wad added and then500 μl (2.2 nmol) of the purified hg-Gaussia luciferase was added toinitiate a modification reaction. The reaction was conducted at 25° C.overnight in the dark. A cysteine solution was added thereto to a finalconcentration of 0.2 mM. The mixture was allowed to stand at roomtemperature for 30 minutes to inactivate the unreactedmaleimide-activated biotin. The inactivated maleimide biotin reagent wasremoved using an Amicon Ultra column (manufactured by Millipore). Thebiotinylated hg-Gaussia luciferase was thus prepared.

The luminescence activity was compared between the biotinylatedhg-Gaussia luciferase obtained as described above and hg-Gaussialuciferase before the reaction. As a result, there is no significantloss on the luminescence activity due to biotinylation; the biotinylatedhg-Gaussia luciferase retained the luminescence activity of 98% or more.

Example 8 Correlation Between Protein Concentration and LuminescenceActivity of Biotinylated hg-Gaussia Luciferase

In order to use the biotinylated hg-Gaussia luciferase as a detectionprobe, it is required that the protein concentration and theluminescence activity are in a linear correlation. The biotinylatedhg-Gaussia luciferase was diluted to a concentration in a range from 100femtograms to 1 nanogram, 100 μl of substrate coelenterazine (0.5 ng/μl)was injected, and the luminescence activity was measured on an apparatusfor measuring luminescence Centro LB960 (manufactured by Berthold). Thecorrelation between the maximum luminescence intensity (Imax) and theprotein concentration is shown in FIG. 7. The linear correlation wasobserved between the luminescence intensity and biotinylated hg-Gaussialuciferase. The results reveal that the amount of biotinylatedhg-Gaussia luciferase can be quantitatively determined by luminescence.

Example 9 Luminescent Immunoassay of α-Fetoprotein (AFP) UsingBiotinylated hg-Gaussia Luciferase 1) Coating of Anti-FetoproteinAntibody (Anti-AFP Antibody)

The anti-AFP antibody (manufactured by Japan Clinical Laboratories,Inc., Clone No. 6D2, subclass IgG2a-κ, hereinafter referred to as “6D2”)was prepared at a concentration of 5 μg/ml, using 50 mM carbonate buffersolution (pH 9.6) containing 0.05% sodium azide. The solution wasdispensed into a 96-well microplate (manufactured by Nunc, #437796) inan amount of 100 μl/well and allowed to stand at room temperatureovernight for coating. After the standing, the carbonate buffer solutionwas withdrawn, a solution of 150 mM NaCl (manufactured by Wako PureChemical Industry) and 20 mM Tris-HCl (manufactured by Wako PureChemical Industry) (hereinafter referred to as TBS) containing 1% bovineserum albumin (manufactured by Sigma, hereinafter referred to as BSA), 2mM EDTA (EDTA.2Na, manufactured by Dojin Chemical Laboratory) and 0.05%sodium azide (manufactured by Wako Pure Chemical Industry) (hereinafterreferred to as a post-coating solution) was dispensed in an amount of200 μl/well, which was then allowed to stand at 4° C. overnight.

2) Detection of AFP by Biotinylated hg-Gaussia Luciferase

After the standing, the post-coating solution was removed. The wellswere washed 3 times with 340 μl of TBS containing 0.01% Tween 20 and 2mM EDTA (referred to as TBST-E), α-fetoprotein (manufactured by Dako,abbreviated as AFP), which was diluted from 0.0125 ng/ml to 125 ng/mlwith TBS containing 10% Block Ace (manufactured by Snow Brand MilkProducts) and 2 mM EDTA, was dispensed into a 96-well microplate in anamount of 50 μl/well. The biotinylated anti-AFP antibody (manufacturedby Japan Clinical Laboratories, Inc., Clone No. 1D5, subclass IgG1-κ,hereinafter referred to as “1D5”) diluted to 74.5 ng/ml, was furtherdispensed in an amount of 50 μl/well, which was then allowed to stand at30° C. for an hour. The reaction solution was withdrawn from the platedescribed above and the wells were washed 3 times with 340 μl of TBST-E.Next, 50 μl of streptavidin diluted to 200 pmol/ml with PBS containing10% Block Ace, 0.01% Tween 20 (manufactured by Bio-Rad) and 10 mM EDTA(hereinafter referred to as PBSE-TB) was mixed with 50 μl of thebiotinylated hg-Gaussia luciferase diluted to 400 pmol/ml. The mixturewas reacted at room temperature for 30 minutes. Thereafter, the solutiondiluted to 80-fold with PBSE-TB was dispensed onto the plate above in anamount of 100 μl/well. The mixture was allowed to stand at 30° C. for 30minutes. The reaction solution was removed and the wells were washed 3times with 340 μl of TBST-E. Then, substrate coelenterazine diluted withPBST-E to 0.5 ng/μl was injected into the wells in an amount of 100μl/well. The luminescence intensity was measured on an apparatus formeasuring luminescence Centro LB960 (manufactured by Berthold) for 10seconds in 0.1 second intervals to determine the maximum luminescenceintensity (Imax). The standard curve of AFP obtained from the Imax andAFP concentration is shown in FIG. 8.

Example 10 Preparation of Biotinylated hgA-Gaussia Luciferase byMaleimide-Activated Biotin

To 650 μl of PBS-E, 4.2 μl (4.2 nmol) of maleimide-activated biotin(manufactured by Pierce, EZ-Link PEO-Maleimide-Activated Biotin; spacerlength: 29.1 angstroms) adjusted to 1 mM with PBS-E, wad added and then350 μl (2.1 nmol) of the purified hgA-Gaussia luciferase was added toinitiate a modification reaction. The reaction was carried out at 25° C.for 2 hours in the dark. A cysteine solution was added thereto to afinal concentration of 0.2 mM. The mixture was allowed to stand at roomtemperature for 30 minutes to inactivate the unreactedmaleimide-activated biotin. The inactivated maleimide biotin reagent wasremoved using an Amicon Ultra column (manufactured by Millipore). Thebiotinylated hgA-Gaussia luciferase was thus prepared.

The luminescence activity was compared between the biotinylatedhgA-Gaussia luciferase obtained as described above and hgA-Gaussialuciferase before the reaction. As a result, there is no significanteffect on the luminescence activity due to biotinylation and thebiotinylated hgA-Gaussia luciferase retained the luminescence activityof 98% or more.

Example 11 Quantitative Property of Biotinylated hgA-Gaussia Luciferase

In order to use the biotinylated hgA-Gaussia luciferase as a detectionprobe, it is required that the protein concentration and theluminescence activity are in a linear correlation. The biotinylatedhgA-Gaussia luciferase mutant was diluted to a concentration in a rangefrom 200 femtograms to 200 picograms, and 100 μl of substratecoelenterazine (0.5 ng/μl) was injected therein. The luminescenceactivity was measured with an apparatus for measuring luminescenceCentro LB960 (manufactured by Berthold). The correlation between themaximum luminescence intensity (Imax) and the protein concentration isshown in FIG. 9. The linear correlation was observed between theluminescence intensity and the biotinylated hgA-Gaussia luciferase. Theresults reveal that the amount of the biotinylated hgA-Gaussialuciferase can be quantitatively determined by luminescence.

Example 12 Luminescent Immunoassay of α-Fetoprotein (AFP) UsingBiotinylated hg-Gaussia Luciferase 1) Coating of Anti-FetoproteinAntibody (Anti-AFP Antibody)

The anti-AFP antibody (6D2) was prepared in a concentration of 5 μg/ml,using 50 mM carbonate buffer solution (pH 9.6) containing 0.05% sodiumazide. The solution was dispensed into a 96-well microplate(manufactured by Nunc, #437796) in an amount of 100 μl/well and allowedto stand at room temperature overnight for coating. After the standing,the carbonate buffer solution was withdrawn and the post-coatingsolution was dispensed in an amount of 200 μl/well, which was thenallowed to stand at 4° C. overnight.

2) Detection of AFP by Biotinylated hgA-Gaussia Luciferase

After the standing, the post-coating solution was removed. The wellswere washed 3 times with 340 μl of TBST-E. AFP, which was diluted in arange from 0.0125 ng/ml to 125 ng/ml with TBS containing 10% Block Aceand 2 mM EDTA, was dispensed into a 96-well microplate in an amount of50 μl/well. The biotinylated anti-AFP antibody (1D5) diluted to 74.5ng/ml was further dispensed in an amount of 50 μl/well, which was thenallowed to stand at 30° C. for an hour. The reaction solution waswithdrawn from the plate described above and the wells were washed 3times with 340 μl of TBST-E. Next, 50 μl of streptavidin diluted to 200pmol/ml with PBSE-TB was mixed with 50 μl of the biotinylatedhgA-Gaussia luciferase diluted to 400 pmol/ml. The mixture was reactedat room temperature for 30 minutes. Thereafter, the solution diluted to80-fold with PBSE-TB was dispensed onto the plate above in an amount of100 μl/well, followed by allowing to stand at 30° C. for 30 minutes. Thereaction solution was removed and the wells were washed 3 times with 340μl of TBST-E. Then, substrate coelenterazine diluted to 0.5 ng/μl withPBST-E was injected into the wells in an amount of 100 μl/well. Theluminescence intensity was measured on an apparatus for measuringluminescence Centro LB960 for 10 seconds in 0.1 second intervals todetermine the maximum luminescence intensity (Imax). The standard curveof AFP obtained from the Imax and AFP concentration is shown in FIG. 10.

Example 13 Preparation of hgA-Gaussia Luciferase-Labeled Antibody

1) Preparation of Sulfhydryl Reactive Antibody (Maleimidated Antibody)(Conjugation of Anti-AFP Antibody and Crosslinking Reagent havingMaleimide Group)

To a solution of 50 μg (0.3 nmol) of the anti-AFP antibody (manufacturedby Japan Clinical Laboratories, Inc., Clone No. 1D5, subclass IgG1-κ,hereinafter referred to as “1D5”) in 78.3 μl of 66 mM phosphate bufferedsaline containing 0.1 mM EDTA (pH 7.4, hereinafter referred to as“buffer A”), 3 μl of sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, PIERCE) wasadded (sulfo-SMCC, 3 nmol, 1D5/sulfo-SMCC=1/10). The mixture was reactedat 4° C. for 2 hours. After completion of the reaction, 1 (10 nmol) of10 mM glycine was added to the reaction mixture and allowed to stand at25° C. for at least 10 minutes to stop the reaction. The inactivatedsulfo-SMCC was removed by centrifugation (manufactured by Hitachi,CR20B2) at 4° C. and 6,000 rpm for 15 minutes using an Amicon Ultra-4Spin Column (manufactured by Millipore; molecular weight cutoff, 10,000)to give the conjugate of anti-AFP antibody and the crosslinking reagenthaving the maleimide group (hereinafter sometimes referred to as“maleimidated 1D5”). The recovery rate of the maleimidated 1D5 ascalculated from E^(1%) _(280 nm)=14 was 70.6% (0.24 nmol, 35.3 μg).

2) Reaction of hgA-GL with Sulfhydryl Reactive Antibody (Maleimidated1D5)

To 0.24 nmol of the maleimidated 1D5, 0.72 nmol of the purifiedhgA-Gaussia luciferase (hereinafter sometimes referred to as “hgA-GL”)was added. The mixture was reacted at 4° C. overnight. After completionof the reaction, 1 (10 nmol) of 10 mM cysteine aqueous solution wasadded to the reaction mixture to give the conjugate of hgA-GL and themaleimidated 1D5 (hereinafter sometimes referred to as “hgA-GL-Ab1D5”).

The hgA-GL-Ab1D5 retained the luminescence activity of 96%, withoutsignificant loss of the luminescence activity. The hgA-GL-Ab1D5 obtainedwas applied to SDS-PAGE analysis on a 12% separation gel under reducingconditions. As a result, a band was detected at 84 kDa, indicating theformation of conjugate of hgA-GL (29 kDa) with a heavy chain (55 kDa) of1D5.

Example 14 Quantitative Property of hgA-GL-Ab1D5

In order to use hgA-GL-Ab1D5 as a detection probe, it is required thatthe protein concentration and the luminescence activity are in a linearcorrelation. The concentration of hgA-GL-Ab1D5 was diluted in a rangefrom 3 picograms to 3,000 picograms, 100 μl of substrate coelenterazine(0.5 ng/μl) was injected, and the luminescence activity was measuredwith an apparatus for measuring luminescence Centro LB960 (manufacturedby Berthold). The correlation between the maximum luminescence intensity(Imax) and the protein concentration is shown in FIG. 11. The linearcorrelation was observed between the luminescence intensity andhgA-GL-Ab1D5. The results reveal that the amount of hgA-GL-Ab1D5 can bequantitatively determined by luminescence.

Example 15 Luminescent Immunoassay of AFP Using hgA-GL-Ab1D5 1) Coatingof Anti-AFP Antibody

The anti-AFP antibody (Clone No. 6D2, subclass IgG2a-κ, manufactured byJapan Clinical Laboratories, Inc., hereinafter sometimes referred to as“6D2”) was prepared in a concentration of 5 μg/ml, using 50 mM carbonatebuffer solution (pH 9.6) containing 0.05% sodium azide. The solution wasdispensed into a 96-well microplate (manufactured by Nunc, #437796) inan amount of 100 μl/well and allowed to stand at 25° C. overnight forcoating.

2) Post-Coating

After the standing, the carbonate buffer solution was withdrawn and asolution of 150 mM NaCl (Wako Pure Chemical Industry) and 20 mM Tris-HCl(Wako Pure Chemical Industry) (hereinafter referred to as TBS)containing 1% bovine serum albumin (Sigma), 2 mM EDTA (EDTA.2Na, DojinChemical Laboratory) and 0.05% sodium azide (Wako Pure ChemicalIndustry) (hereinafter referred to as a post-coating solution) wasdispensed in an amount of 200 μl/well, which was then allowed to standat 4° C. overnight.

3) Reaction of AFP with hgA-GL-Ab1D5

The post-coating solution was removed. After the wells were washed 3times with 340 μl of TBST-E, 110 μg/ml of AFP (Dako) was diluted in TBScontaining 10% Block Ace (Dainippon Pharmaceutical) and 5 mM EDTA(hereinafter referred to as diluent) to prepare a serial dilution from10 pg/ml to 200 ng/ml. The dilution was dispensed in an amount of 50μl/well. Furthermore, hgA-GL-Ab1D5 diluted to 74.5 ng/ml with thediluent was dispensed in an amount of 50 μl/well, which was then allowedto stand at 25° C. for an hour.

4) Measurement of Luminescence

After the standing, the reaction solution was removed and the wells werewashed 3 times with 340 μl of TBST-E. Then, substrate coelenterazinediluted to 0.5 ng/μl with PBST-E was injected into the wells in anamount of 100 μl/well. The luminescence intensity was measured on anapparatus for measuring luminescence Centro LB960 (manufactured byBerthold) for 10 seconds in 0.1 second intervals to determine themaximum luminescence intensity (Imax).

The standard curve of AFP obtained by bioluminescent immunoassay usinghgA-GL-Ab1D5 is shown in FIG. 12. The results revealed that thedetection limit of AFP was 4×10⁻² ng/ml and the dynamic range was 4×10⁻²to 10² ng/ml, indicating that hgA-GL-Ab1D5 is excellent forimmunoassays.

Example 16 Preparation of Fluorescence-Labeled Gaussia Luciferase

After 2.2 nmol of purified hg-Gaussia luciferase was dissolved in 1 mlof 10 mM EDTA-containing PBS (manufactured by Sigma; 0.137 M sodiumchloride and 0.0027 M potassium chloride, pH 7.4), the mixture was addedto 22 nmol of a solution of fluorescein-5-maleimide (manufactured byPierce) in dimethylformamide. The mixture was then reacted at 4° C. for16 hours. The reaction solution was centrifuged at 4° C. and 6,000 rpmfor 10 minutes using an Amicon Ultra-4 Spin Column (manufactured byMillipore; molecular weight cutoff, 10,000). After concentration, thecolumn was washed twice with 2 ml of PBS solution containing 10 mM EDTAand 0.01% Tween 20 and the total volume was made 1 ml. The recovery rateof the fluorescence-labeled Gaussia luciferase activity was 92%.

Example 17 Bioluminescence Resonance Energy Transfer (BRET) byFluorescence-Labeled Gaussia Luciferase

After 5 μg of coelenterazine dissolved in 5 μl of ethanol was added to0.99 ml of PBS solution containing 10 mM EDTA and 0.01% Tween 20, 5 μlof the fluorescence-labeled Gaussia luciferase (1 μg) was added to themixture to initiate a luminescence reaction. At the same time,luminescence emission spectra were measured on a Jasco FP-6500(manufactured by JASCO Corporation) with the excitation light sourceturned off. The measurement was performed under the conditions: emissionband width, 20 nm; response, 0.2 second; scan speed, 2000 nm/min at 22to 25° C., using a quartz cuvette (10 mm light path). The results of themeasured spectra are shown in FIG. 13. Light emission by Gaussialuciferase alone exhibits only the maximum at 467 nm; with thefluorescence-labeled Gaussia luciferase, a new peak is formed at 510 nm.This is because the luminescence energy produced as a result of theoxidation of coelenterazine by Gaussia luciferase occurs as a result ofthe energy transfer to a fluorescent dye fluorescein bound to Gaussialuciferase. The energy transfer efficiency is estimated to beapproximately 50%.

Reference Example 1 Construction of Gaussia Luciferase-Apoaequorin-S142CExpression Vector

The Gaussia luciferase-apoaequorin-S142C fused gene expression vectorhaving the Gaussia luciferase gene and the apoaequorin-S142C genewherein the serine residue 142 of apoaequorin was replaced with cysteineresidues was constructed as follows.

The Gaussia luciferase gene (hGL gene) was prepared from Gaussialuciferase gene-bearing pcDNA3-hGL (manufactured by LUX) by PCR. Theapoaequorin-S142C gene wherein the serine residue 142 of apoaequorin wasreplaced with cysteine residues was prepared by PCR using pAM-HEobtained by subcloning the HindIII-EcoRI fragment of apoaequoringene-bearing pAQ440 (cf., JPA 61-135586) into pUC9 vector. pCold II(Takara-Bio) was used as the expression vector.

The serine residue 142 of apoaequorin was replaced with cysteineresidues according to the following procedures. Using pAM-HE as atemplate, PCR was performed (cycle conditions, 25 cycles; 1 min/94° C.,1 min/50° C. and 1 min/72° C.) with a PCR kit (manufactured byTakara-Bio) using the following two primers: AQ-20N/XhoI and AQ-S142C—R,to amplify the desired DNA region.

AQ-20N/XhoI (SEQ ID NO: 21) (5′ccg CTC GAG ACA TCA GAC TTC GAC AAC CCA 3′;the XhoI restriction enzyme site underlined), AQ-S142C-R (SEQ ID NO: 22)(5′ ATC TTC GCA TGA TTG GAT GAT 3′).

Similarly, PCR was performed (cycle conditions, 25 cycles; 1 min/94° C.,1 min/50° C. and 1 min/72° C.) with a PCR kit (manufactured byTakara-Bio) using the following two primers: AQ-S142C-F and AEQ-C-PstI,to amplify the desired DNA region.

AQ-S142C-F (SEQ ID NO: 23) (5′ CAA TCA TGC GAA GAT TGC GAG 3′),AEQ-C-PstI (SEQ ID NO: 24) (5′cgg CTG CAG TTA GGG GAC AGC TCC ACC GTA GAGCTT 3′; the PstI restriction enzyme site underlined)

Using the two PCR products obtained as templates, PCR was performed(cycle conditions, 25 cycles; 1 min/94° C., 1 min/50° C. and 1 min/72°C.) with a PCR kit (manufactured by Takara-Bio) using PCR primers:AQ-20N/XhoI (SEQ ID NO: 21) and AEQ-C-PstI (SEQ ID NO: 24) to give theapoaequorin gene with the cysteine residues replaced for the serineresidue 142. The fragment obtained was purified by a PCR purificationkit (manufactured by Qiagen). After digestion with restriction enzymesXhoI/PstI in a conventional manner, the fragment was ligated topBluescript II SK (+) (Stratagene) at the restriction enzyme XhoI/PstIsites to construct the vector pBlue-AQ-S142C.

Subsequently, PCR was performed (cycle conditions, 25 cycles; 1 min/94°C., 1 min/50° C. and 1 min/72° C.) for the Gaussia gene with a PCR kit(manufactured by Takara-Bio) using pcDNA3-hGL (manufactured by ProlumeLtd.) as a template and using the following two primers:GL-25N/Kpn-EcoRI and GL-24N-TAA/XhoI to amplify the Gaussia gene.

GL-25N/Kpn-EcoRI (5′ ggc GGT ACC GAA TTC AAG CCC ACC GAG AAC AAC 3′; theAsp718I restriction enzyme site underlined) (SEQ ID NO: 25),

GL-24N-TAA/XhoI (5′ ccg CTC GAG GTC ACC ACC GGC CCC CTT GAT 3′; the XhoIrestriction enzyme site underlined) (SEQ ID NO: 26)

The fragment obtained was purified by a PCR Purification Kit(manufactured by Qiagen). After digestion with restriction enzymesAsp718I/XhoI in a conventional manner, the fragment was ligated topBlue-AQ-S142C at the restriction enzyme Asp718I/XhoI sites to constructthe vector pBlue-GL-AQ-S142C.

The vector pBlue-GL-AQ-S142C was digested with restriction enzymesEcoRI/PstI in a conventional manner and then ligated to pColdII at therestriction enzyme EcoRI/PstI sites to construct the expression vectorpCold-GL-AQ-S142C (FIG. 14). The insert DNA was confirmed by nucleotidesequencing on a DNA Sequencer (manufactured by ABI).

The nucleotide sequence of the insert DNA is shown by SEQ ID NO: 27, andthe amino acid sequence of a fusion protein encoded by the insert DNA isshown by SEQ ID NO: 28.

Reference Example 2 Purification of Recombinant GaussiaLuciferase-Aequorin-S142C Fusion Protein

The recombinant Gaussia luciferase-aequorin-S142C fusion protein wasobtained as follows. The recombinant Gaussia luciferase-aequorin-S142Cwas expressed in E. coli using the expression vector pCold-GL-AQ-S142Cand purified by nickel-chelate column chromatography, whereby theapoaequorin part is regenerated to give the recombinant Gaussialuciferase-aequorin-S142C.

1) Expression of Recombinant Gaussia Luciferase-Apoaequorin-S142C in E.Coli

The expression vector pCold-GL-AQ-S142C for the Gaussialuciferase-apoaequorin-S142C gene was used to express the recombinantGaussia luciferase-apoaequorin-S142C in E. coli. This vector wasintroduced into the E. coli BL21 strain in a conventional manner. Thetransformant obtained was inoculated in 10 ml of LB liquid medium (10 gof bactotryptone, 5 g of yeast extract and 5 g of sodium chloride per 1liter of water, pH 7.2) containing ampicillin (50 μg/ml), followed byincubation at 37° C. for 18 hours. Next, the culture broth was added to400 ml×5 tubes of fresh LB liquid medium (2 L in total) and incubated at37° C. for 5 hours. After cooling in an ice water,isopropyl-β-D(−)-thiogalactopyranoside (IPTG, manufactured by Wako PureChemical Industry) was added to the culture to a final concentration of0.2 mM, followed by incubation at 15° C. for further 17 hours. Aftercompletion of the incubation, the cells were recovered by centrifugation(5,000 rpm, 5 mins.) and provided for use as the starting material forprotein extraction.

2) Extraction of Gaussia Luciferase-Apoaequorin-S142C from CulturedCells

The cultured cells collected were suspended in 100 ml of 50 mM Tris-HCl(pH 7.6) and disrupted by ultrasonication (manufactured by Branson,Sonifier Model Cycle 250) 3 times each for 3 minutes under ice cooling.The cell lysate was centrifuged at 10,000 rpm (12,000×g) at 4° C. for 20minutes to give soluble fractions. The resultant soluble fractions weresuspended in 100 ml of 50 mM Tris-HCl (pH 7.6) containing 6 M urea. Thesuspension was subjected to ultrasonic treatment under ice cooling,followed by centrifugation at 10,000 rpm (12,000×g) and 4° C. for 10minutes. The urea soluble fraction obtained was used as the startingmaterial for purification of the Gaussia luciferase-apoaequorin-S142C.

3) Purification of Recombinant Gaussia Luciferase-Apoaequorin-S142C fromthe Urea Soluble Fraction

The recombinant expression protein has 6 histidine sequences at theamino terminus and can be purified by affinity chromatography on anickel-chelate gel.

First, the 6M urea soluble fraction was applied on a nickel-chelatecolumn (Amersham Bioscience, column size: diameter 2.5×6 cm)equilibrated with 50 mM Tris-HCl (pH 7.6) containing 6M urea to adsorbthe Gaussia luciferase-apoaequorin-S142C. The column adsorbed with theGaussia luciferase-apoaequorin-S142C was washed with 150 ml of 50 mMTris-HCl (pH 7.6) containing 6M urea. The Gaussialuciferase-apoaequorin-S142C was then eluted by 50 mM Tris-HCl (pH 7.6)containing 6M urea and 0.1M imidazole (manufactured by Wako PureChemical Industry). Protein concentration was determined by the methodof Bradford using a commercially available kit (manufactured by Bio-Rad)and bovine serum albumin (manufactured by Pierce) as a standard. From 2L of the cultured cells, 106 mg of the Gaussialuciferase-apoaequorin-S142C was obtained.

4) Preparation of Gaussia Luciferase-aequorin-S142C from GaussiaLuciferase-Apoaequorin-S142C

Regeneration of the Gaussia luciferase-aequorin-S142C from Gaussialuciferase-apoaequorin-S142C was carried out as follows. First, theGaussia luciferase-apoaequorin-S142C was treated with a reducing agent,2-mercaptoethanol, and then contacted with a luminescent substrate(coelenterazine) to regenerate aequorin. Thereafter, the reducing agentwas removed by a dialysis treatment. Gaussia luciferase was convertedinto its active form by refolding to give the Gaussialuciferase-aequorin-S142C protein having the Gaussia luciferase activityand the aequorin activity.

Specifically, the Gaussia luciferase-apoaequorin-S142C solution (50μg/10 eluted by 0.1 M imidazole and 6 M urea from the nickel-chelatecolumn was dissolved in 990 μl of 30 mM Tris-HCl (pH 7.6) containing 10mM EDTA. The solution was mixed with 2-mercaptoethanol in a finalconcentration of 0.35% (v/v). After the mixture was allowed to stand at37° C. for 30 minutes, it was confirmed that the luminescence activityof Gaussia luciferase was inactivated. A solution of 5 μg of substratecoelenterazine (1 μg/μl) in ethanol was added and a reaction forregeneration of aequorin was carried out at 4° C. overnight. Next, thesolution of the regenerated aequorin obtained was dialyzed at 4° C.overnight with 4 L of 100 mM ammonium carbonate solution (pH 8.0)containing 10 mM EDTA to give the recombinant Gaussialuciferase-aequorin-S142C protein (40 μg).

Example 18 Comparison of Luminescence Activity

With respect to hg-Gaussia luciferase obtained by the method describedin EXAMPLE 5 3) above, hgA-Gaussia luciferase obtained by the methoddescribed in EXAMPLE 6 3) above and the recombinant Gaussialuciferase-aequorin-S142C protein obtained by the method described inREFERENCE EXAMPLE 2 4) above, the luminescence activity was comparedbetween the respective fusion proteins. Specifically, the luminescenceactivity between the respective fusion proteins was compared accordingto modifications of the method described in EXAMPLE 5 5). The resultsare shown in TABLE 3 below.

From TABLE 3, it is found that hg-Gaussia luciferase and hgA-Gaussialuciferase in which the number of amino acid residues in the portionbetween the first region and the C terminus except for the first regionis 21 to 36 residues show the specific luminescence activity higher byapproximately 500 times than the recombinant Gaussialuciferase-aequorin-S142C protein in which the number of amino acidresidues in the portion between the first region and the C terminusexcept for the first region is 188 residues.

TABLE 3 Comparison of Luminescence Activity between Respective FusionProteins Number of amino Number of Number of acids in the portioncysteines in the Number of amino between the first first region/Specific amino acids acids in region and the C number of luminescence inthe first the second terminus except for cysteines in the activityFusion protein region region the first region second region (10⁷ rlu/mg)Hg-Gaussia 168 15 21 10/1 1055 luciferase (2* + 15 + 4*) hgA-Gaussia 16815 36 10/1 957 luciferase (2* + 15 + 2* + 15 + 2*) Recombinant 168 186188 10/4 2 Gaussia luciferase- (2* + 186) aequorin-S142C proteinSymbol * denotes linker sequences for the restriction enzyme sites.

Based on EXAMPLES above, it is found that the luciferase (fusionprotein) of the invention exhibits a high catalytic ability for aluminescence activity. It is also found that, since the luciferase iscapable of binding to other useful substances (fluorescent substances;ligands such as biotin, antibodies, etc.) via the thiol group ofcysteines introduced into the luciferase of the invention, theluciferase can be utilized for the bioluminescence energy transfer orthe detection of substances specific to ligands.

Sequence Listing Free Text SEQ ID NO: 1

DNA sequence of the expression vector pPICZα-hgLinker having a hingesequence and a multicloning site, prepared in EXAMPLE 1

SEQ ID NO: 2

Amino acid sequence of the protein encoded by the DNA sequence of theexpression vector pPICZα-hgLinker having a hinge sequence and amulticloning site, prepared in EXAMPLE 1

SEQ ID NO: 3

DNA sequence encoding the hg-Gaussia luciferase fusion protein insertedinto the expression vector pPICZα-hgGL-H prepared in EXAMPLE 2

SEQ ID NO: 4

Amino acid sequence of the hg-Gaussia luciferase fusion protein insertedinto the expression vector pPICZα-hgGL-H prepared in EXAMPLE 2

SEQ ID NO: 5

DNA sequence encoding the hg-Gaussia luciferase fusion protein insertedinto the expression vector pCold-hgGL prepared in EXAMPLE 3

SEQ ID NO: 6

Amino acid sequence of the hg-Gaussia luciferase fusion protein insertedinto the expression vector pCold-hgGL prepared in EXAMPLE 3

SEQ ID NO: 7

DNA sequence encoding the hgA-Gaussia luciferase fusion protein insertedinto the expression vector pCold-hgA-GL prepared in EXAMPLE 4

SEQ ID NO: 8

Amino acid sequence of the hgA-Gaussia luciferase fusion proteininserted into the expression vector pCold-hgA-GL prepared in EXAMPLE 4

SEQ ID NO: 9

Nucleotide sequence of the primer used in EXAMPLE 1

SEQ ID NO: 10

Nucleotide sequence of the primer used in EXAMPLE 1

SEQ ID NO: 11

Nucleotide sequence of the primer used in EXAMPLE 1

SEQ ID NO: 12

Nucleotide sequence of the primer used in EXAMPLE 1

SEQ ID NO: 13

Nucleotide sequence of the primer used in EXAMPLE 2

SEQ ID NO: 14

Nucleotide sequence of the primer used in EXAMPLE 2

SEQ ID NO: 15

Nucleotide sequence of the primer used in EXAMPLE 4

SEQ ID NO: 16

Nucleotide sequence of the primer used in EXAMPLE 4

SEQ ID NO: 17

DNA sequence encoding the catalytic domain of Gaussia luciferase

SEQ ID NO: 18

Amino acid sequence of the catalytic domain of Gaussia luciferase

SEQ ID NO: 19

DNA sequence encoding the hinge

SEQ ID NO: 20

Amino acid sequence of the hinge

SEQ ID NO: 21

Nucleotide sequence of the primer used in REFERENCE EXAMPLE 1

SEQ ID NO: 22

Nucleotide sequence of the primer used in REFERENCE EXAMPLE 1

SEQ ID NO: 23

Nucleotide sequence of the primer used in REFERENCE EXAMPLE 1

SEQ ID NO: 24

Nucleotide sequence of the primer used in REFERENCE EXAMPLE 1

SEQ ID NO: 25

Nucleotide sequence of the primer used in REFERENCE EXAMPLE 1

SEQ ID NO: 26

Nucleotide sequence of the primer used in REFERENCE EXAMPLE 1

SEQ ID NO: 27

DNA sequence encoding the Gaussia luciferase-apoaequorin-S142C fusionprotein, inserted into the expression vector pCold-GL-AQ-S142 preparedin REFERENCE EXAMPLE 1

SEQ ID NO: 28

Amino acid sequence of the Gaussia luciferase-apoaequorin-S142C fusionprotein, inserted into the expression vector pCold-GL-AQ-S142 preparedin REFERENCE EXAMPLE 1

1-27. (canceled)
 28. A method for determining a substance specific to aligand, which comprises using a complex comprising the fusion proteincomprising: (1) a first region selected from the group consisting of (a)to (d) below: (a) a region consisting of the amino acid sequence of SEQID NO: 18; (b) a region consisting of the amino acid sequence of SEQ IDNO: 18 wherein 1 to 10 amino acids are deleted, substituted, insertedand/or added and having a catalytic ability for a luminescence activitywith a luciferin which is a substrate; (c) a region consisting of anamino acid sequence having at least 90% homology to the amino acidsequence of SEQ ID NO: 18 and having a catalytic ability for aluminescence activity with a luciferin which is a substrate; and, (d) aregion consisting of an amino acid sequence encoded by a polynucleotidewhich hybridizes under high stringent conditions to a polynucleotideconsisting of a nucleotide sequence complementary to the nucleotidesequence of SEQ ID NO: 17 and having a catalytic ability for aluminescence activity with a luciferin which is a substrate, wherein thehigh stringent conditions are 5×SSC, 5×Denhardt's solution, 0.5% (w/v)SDS, 50% (v/v) formamide and 50° C.; and, (2) a second region consistingof an amino acid sequence for a polypeptide having at least one cysteineresidue for binding to other useful compound via its thiol group,wherein the second region is selected from the group consisting of (e)to (h) below: (e) a region consisting of the amino acid sequence of SEQID NO: 20; (f) a region comprising the amino acid sequence of SEQ ID NO:20 wherein 1 to 3 amino acids are deleted, substituted, inserted and/oradded and having at least one cysteine residue for binding to otheruseful compound via the thiol group; (g) a region comprising an aminoacid sequence having at least 90% homology to the amino acid sequence ofSEQ ID NO: 20 and having at least one cysteine residue for binding toother useful compound via the thiol group; and, (h) a region comprisingan amino acid sequence encoded by a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 19 and having at least one cysteine residue for binding to otheruseful compound via the thiol group, wherein the high stringentconditions are 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v)formamide and 50° C.; and the ligand bound to the fusion protein via thethiol group of the cysteine residue in the second region.
 29. The methodaccording to claim 28, wherein: (1) the first region is a regionconsisting of the amino acid sequence of SEQ ID NO: 18, and, (2) thesecond region is a region consisting of the amino acid sequence of SEQID NO:
 20. 30. The method according to claim 28, wherein the fusionprotein further comprises an amino acid sequence for promotingtranslation and/or an amino acid sequence for purification.
 31. Themethod according to claim 28, wherein the fusion protein consisting ofan amino acid sequence of SEQ ID NO: 4, 6 or 8.